Ref.

Ares(2020)1224192 - 27/02/2020

D7.4 Analytical Handbook for Microalgae-Based

Novel High Added-Value Products for Cosmetic and
Aquaculture
Algae4A-B - Algae for Aquaculture and Beauty
Development of microalgae-based novel high added-value products for the cosmetic and aquaculture industry
Project nr: 691102 Call reference: H2020-MSCA-RISE-15
st
Start date: 1 January 2016 Duration: 48 months
Analytical Handbook
Deliverable identification
Leading beneficiary:
Related WP: 7 Planned delivery date: M48
Related period: - Actual delivery date: M50
Dissemination level: PU Delivery status: Submitted

Contributors:
Beneficiary Name(s)

Centre National de la Recherche Scientifique William Helbert

Fitoplancton Marino, S.L. Carlos Infante - Lalia Mantecon

Apivita Kallyntika Diaititika Farmaka Anonymi
Emporiki Kai Viotechnikietaireia
Sophia Letsiou
Instituto Andaluz De Investigacion y Formacion Manuel Manchado - Carlos Carballo
Agraria Pesquera Alimentaria Israel Guerrero-Cozar - Raul Navarro
Deborah M. Power - Liliana Anjos - João CR
Centro De Ciencias Do Mar Do Algarve
Cardoso - Rute C. Félix - Patricia I. Pinto
ADM Lifesequencing Juan F. Martinez-Blanch

Lyon Ingénierie Projets Patricia Odet

Reviews
Version Reviewer Date
V1 – V5 All partners January 2020

V6 William Helbert 27/02/2020

–1–
 Analytical Handbook for Microalgae-Based Novel
High Added-Value Products for Cosmetics and
Aquaculture

Deborah M Power1, Liliana Anjos1, Manuel Manchado2, Sophia Letsiou3, João CR

Cardoso1, Rute C Félix1, Patricia I Pinto1, Carlos Infante4, Carlos Carballo2, Lalia
Mantecon4, Raul Navarro2, Israel Guerrero-Cozar2, Juan F. Martinez-Blanch5,
William Helbert6

1
Centro de Ciencias do Mar, Universidade do Algarve, 8005-139, Faro, Portugal
2
Instituto Andaluz De Investigacion y Formacion Agraria Pesquera Alimentaria (IFAPA), Centro El
Toruño, Junta de Andalucía, Camino Tiro Pichón s/n, 11500, El Puerto de Santa María, Spain
3
APIVITA SA, Industrial Park of Markopoulo, 19003, Athens, Greece
4
Fitoplancton Marino, S.L., 11500, El Puerto de Santa María, Spain
5
LifeSequencing, Parc Cientific Universidad De Valencia, Edif. 2, C/ Catedrático Agustín Escardino
Benlloch, 9, 46980, Paterna, Spain
6
Centre National de la Recherche Scientifique, Grenoble, France

Email contacts:
W Helbert: [email protected]
Deborah M Power: [email protected]
Liliana Anjos: [email protected]
João Cardoso: [email protected]
Rute C Félix: [email protected]
Patricia Pinto: [email protected]
Sofia Letsiou: [email protected]
Carlos Carballo: [email protected]
Manuel Manchado: [email protected]
Israel Guerrero-Cozar: [email protected]
Raul Navarro: [email protected]
Carlos Infante: [email protected]
Lalia Mantecon: [email protected]
Juan F. Martinez-Blanch: [email protected]

–2–
 Summary of techniques and methods

CHAPTER 1. MICROALGAL CULTURE ............................................................................................................. 9

1.1 BACKGROUND KNOWLEDGE ................................................................................................................................. 9
1.2 EXPERIMENTAL PROTOCOL FOR SELECTING CULTURE MEDIUM ................................................................................... 10
1.3 MEASURING THE OPTICAL DENSITY (OD) OF CULTURES ............................................................................................ 11
1.4 EXPERIMENTAL PROTOCOL FOR SELECTING THE WAVELENGTH FOR MICROALGAE CULTURE .............................................. 11
1.5 EVALUATING MICROALGAE GROWTH AND METABOLISM UNDER DIFFERENT LIGHT WAVELENGTHS ..................................... 12
1.6 DETERMINING THE EFFECTS OF RED, BLUE AND GREEN LIGHT FILTERING ON THE MICROALGAE METABOLISM ....................... 12
1.7 DETERMINING THE EFFECTS OF RED, BLUE AND GREEN LIGHT FILTERING ON THE MICROALGAE TRANSCRIPTOME................... 13
1.8 ESTABLISHING EXPERIMENTAL FACILITIES FOR INDOOR TRIALS .................................................................................... 13
CHAPTER 2. TRANSFER OF THE OPTIMAL GROWTH CONDITIONS FROM LAB SCALE TO LARGE SCALE
PHOTOBIOREACTORS ................................................................................................................................. 16
2.1 BACKGROUND KNOWLEDGE ............................................................................................................................... 16
2.2 PRODUCTION AT INDUSTRIAL SCALE OF T. CHUII IN OUTDOOR CONDITIONS .................................................................. 16
2.3 DETERMINING GROWTH PERFORMANCE OF INDOOR AND OUTDOOR INDUSTRIAL BIOREACTORS AND IDENTIFYING FACTORS
INFLUENCING MICROALGAE GROWTH ......................................................................................................................... 18
2.4 DETERMINING THE EFFECT OF LIGHT INTENSITY AND TEMPERATURE ON T. CHUII BIOMASS UNDER OUTDOOR CONDITIONS ..... 18
2.5 EXPERIMENTAL ANALYSIS OF MICROALGAE BIOLOGICAL ACTIVITIES OF INTEREST ............................................................ 19
2.5.1 Antioxidant capacity of T. chuii biomass harvested at different times of the day ............................... 19
2.5.2 Superoxide Dismutase (SOD) capacity of T. chuii biomass................................................................... 20
2.6 METABOLOMIC ANALYSIS OF BIOMASS COLLECTED EARLY IN THE MORNING AND MIDDAY................................................ 21
2.6.1. Sample collection ................................................................................................................................ 21
2.6.2 Analysis of the metabolome ................................................................................................................ 21
2.7 INDUSTRIAL SCALE PRODUCTION OF CONTROLLED QUALITY BIOMASS .......................................................................... 22
2.8 UP‐SCALING OF MICROALGAE CULTURES ............................................................................................................... 22
CHAPTER 3. PURIFICATION AND STRUCTURAL ANALYSIS OF MICROALGAE POLYSACCHARIDES ................... 23
3.1 BACKGROUND KNOWLEDGE ............................................................................................................................... 23
3.2 SELECTION OF EPS PRODUCING CYANOBACTERIA STRAINS ........................................................................................ 23
3.3 ANALYTICAL METHODOLOGY .............................................................................................................................. 24
3.3.1 Purification of cyanobacteria EPS ........................................................................................................ 24
3.3.2 Analysis of osidic composition ............................................................................................................. 24
3.3.3 Structural analysis................................................................................................................................ 25
3.3.4 Determination of the protein content of crude microalgae extracts ................................................... 26
CHAPTER 4. PURIFICATION AND ANALYSIS OF MICROALGAE PROTEINS ...................................................... 28
4.1 BACKGROUND KNOWLEDGE ............................................................................................................................... 28
4.2 MICROALGAE TETRASELMIS CHUII TOTAL PROTEIN EXTRACTION USING MECHANICAL DISRUPTION ..................................... 29
4.3 MICROALGAE T. CHUII PROTEOME PROFILE ANALYSIS BY 1D PAGE............................................................................ 31
4.3.1 1D SDS‐PAGE gel preparation .............................................................................................................. 31
4.3.2 1D SDS‐PAGE microalgae protein sample preparation ........................................................................ 32
4.3.3 Electrophoresis .................................................................................................................................... 32
4.3.4 Protein detection................................................................................................................................. 33
4.4 MICROALGAE T. CHUII SAMPLE PREPARATION FOR SWATH QUANTITATIVE PROTEOME ANALYSIS .................................... 33
CHAPTER 5. MOLECULAR METHODS ........................................................................................................... 35
5.1 EXTRACTION OF TOTAL RNA FROM HUMAN AND FISH CELLS ..................................................................................... 35
5.2 SYNTHESIS OF CDNA ........................................................................................................................................ 36
5.3 QUANTITATIVE GENE EXPRESSION: PCR PLATFORM DESIGN AND ANALYSIS .................................................................. 36
5.3.1 Primer design ....................................................................................................................................... 36
5.3.2 Reaction preparation ........................................................................................................................... 36
5.3.3 Data analysis........................................................................................................................................ 36
5.4 GENE EXPRESSION ANALYSIS IN FISH LARVAE AND TISSUES ........................................................................................ 37

–3–
 5.4.1 Total RNA isolation from embryos and larvae ..................................................................................... 37
5.4.2 RT‐qPCR ............................................................................................................................................... 39
5.5 SAMPLING OF BIOLOGICAL SAMPLES FOR MICROBIOME ANALYSES .............................................................................. 40
5.5. EXTRACTION OF TOTAL DNA FOR BACTERIAL MICROBIOME CHARACTERIZATION ........................................................... 40
5.5.1. Lysis of fish samples ............................................................................................................................ 41
5.5.2 Column purification of the DNA ........................................................................................................... 41
5.6 CONSTRUCTION AND SEQUENCING OF MICROBIOME LIBRARIES .................................................................................. 41
5.7 MICROORGANISM‐SPECIFIC QUANTITATIVE PCR .................................................................................................... 42
CHAPTER 6. PROTOCOLS FOR NUTRACEUTICALS ADMINISTRATION ............................................................ 43
6.2 ADMINISTRATION OF MICROALGAL EXTRACTS BY INJECTION ...................................................................................... 43
6.2.1 Preparation of microalgal extract and cytotoxicity test ...................................................................... 43
6.2.2 Fish testing ........................................................................................................................................... 44
6.2.3 Cumulative mortality ........................................................................................................................... 44
6.2.4. Expression profiles in immunological organs of sole treated with chrysolaminarin‐enriched
microalgae extract ........................................................................................................................................ 45
6.3 ADMINISTRATION OF MICROALGAL EXTRACTS BY ORAL INTUBATION............................................................................ 46
6.4 ADMINISTRATION OF MICROALGAL EXTRACTS THROUGH THE DIETS............................................................................. 47
6.4.1 Evaluation of crude extracts of P. tricornutum, yeast ‐glucans and encapsulated microalgal extracts
in fish juveniles.............................................................................................................................................. 47
6.4.2 Evaluation of crude extracts of P. tricornutum and encapsulates in larvae throughout live prey ....... 47
CHAPTER 7. PROTOCOLS FOR FISH EMBRYOS AND LARVAL ASSAYS ............................................................ 49
7.1 BACKGROUND KNOWLEDGE ............................................................................................................................... 49
7.1 FISH EGG DEVELOPMENT AND SPECIES SELECTION ................................................................................................... 49
7.1.1 Evaluate the egg quality ...................................................................................................................... 49
7.1.2 Monitor egg development ................................................................................................................... 49
7.1.3 Criteria for species selection ................................................................................................................ 50
7.2 EMBRYO AND LARVAL SCREENING ASSAYS FOR BIOACTIVE COMPOUNDS ...................................................................... 50
7.2.1 Preparation of microalgae extracts for crude extracts ........................................................................ 50
7.2.2 Preparation of materials for embryos and larval testing ..................................................................... 51
7.2.3. Plate selection and preparation .......................................................................................................... 51
7.2.4 Procedure ............................................................................................................................................. 51
7.2.5 Embryo management .......................................................................................................................... 52
7.3 EXPERIMENTAL DESIGN SETTING .......................................................................................................................... 53
7.4 PROTOCOL FOR MICROSPHERES DELIVERY TO LARVAE .............................................................................................. 53
7.4.1 Live prey enrichment............................................................................................................................ 53
7.4.2 Live prey delivery ................................................................................................................................. 54
7.4.3 Monitoring ........................................................................................................................................... 54
7.5 SURVIVAL ANALYSIS AND SAMPLINGS ................................................................................................................... 54
7.5.1 Biological parameters .......................................................................................................................... 54
7.5.2 Gene expression analysis ..................................................................................................................... 54
7.5.3 Whole‐mount in situ hybridization (WISH) .......................................................................................... 54
7.5.4 Histology .............................................................................................................................................. 54
7.5.5 Microbiome analysis ............................................................................................................................ 55
7.6 LARVAL TESTING .............................................................................................................................................. 55
7.7 HISTOLOGICAL AND STAINING TECHNIQUES ........................................................................................................... 56
7.7.1 Tissue processing and general histology .............................................................................................. 56
7.7.2 Haematoxylin and eosin staining ......................................................................................................... 57
7.7.3 Combination of Alcian Blue, Periodic Acid‐Schiff and Orange G of head kidney ................................. 57
7.7.4 Picro‐Sirius Red of skin ......................................................................................................................... 58
7.7.5 Histomorphometric analysis ................................................................................................................ 58
7.9 WHOLE‐MOUNT IN SITU HYBRIDIZATION (WISH) ................................................................................................... 58
7.9.1 Probe preparation ................................................................................................................................ 58
7.9.2 PCR purification (using a vacuum protocol) ......................................................................................... 59
7.9.3 PCR Cloning .......................................................................................................................................... 59
7.9.4 Sequencing of PCR products ................................................................................................................ 59

–4–
 7.9.5 Riboprobe preparation......................................................................................................................... 59
7.9.6 Hybridization protocols ........................................................................................................................ 60
7.10 ENZYMATIC ANALYSIS OF PROTEASES .................................................................................................................. 61
7.10.1 Reagents ............................................................................................................................................ 62
7.10.2 Procedures ......................................................................................................................................... 62
CHAPTER 8. IN VITRO DERMAL CELL CULTURES AND WOUND HEALING ASSAYS .......................................... 64
8.1 BACKGROUND KNOWLEDGE ............................................................................................................................... 64
8.2 CULTURE OF PRIMARY NORMAL HUMAN DERMAL FIBROBLAST CELLS ........................................................................... 65
8.2.1 Setting up a NHDF cell culture ............................................................................................................. 65
8.2.2 Cell subcultures .................................................................................................................................... 65
8.3 CULTURE OF FISH DERMAL CELLS ......................................................................................................................... 66
8.3.1 Setting up a SJD.1 cell culture .............................................................................................................. 66
8.3.2 Cell subcultures .................................................................................................................................... 67
8.4 IN VITRO SCREENING FOR SKIN BIOACTIVE COMPOUNDS ........................................................................................... 67
8.4.1 MTT assay ............................................................................................................................................ 67
8.4.2 MTT assay using primary teleost cells ................................................................................................. 68
8.4.3 Scratch assay ....................................................................................................................................... 68
8.4.4 Cell migration assay (barrier assay)..................................................................................................... 68
8.4.5 Cell scratch assay ................................................................................................................................. 69
8.4.6 Scratch assay on dermal fish fibroblasts .............................................................................................. 70
8.5. ISOLATION AND PRIMARY CULTURE OF MARINE TELEOST FISH DERMAL CELLS ............................................................... 70
8.5.1 Culture media and conditions .............................................................................................................. 70
8.5.2 Isolation of dermal cells from sea bass ................................................................................................ 71
8.5.3 Skin‐scales explants of juvenile sea bass ............................................................................................. 71
8.5.4 Skin explants of juvenile sea bass ........................................................................................................ 71
8.5.5 Caudal edge fin explants of juvenile sea bass ...................................................................................... 72
8.5.6 Isolation of skin keratinocyte from the scales of adult sea bass .......................................................... 73
CHAPTER 9. REFERENCES ............................................................................................................................ 75

–5–
 General Introduction to the Algae4a-b Handbook
In the context of an MSCA RISE project, Development of Microalgae-based novel high
added-value products for the Cosmetic and Aquaculture industry (Algae4a-b, nº 619902)
substantial scientific and knowledge translation actions were carried out. The consortium
included a microalgae company and a natural cosmetics company along with a sequencing
and diagnostics company and several academic institutes and a technology translation
centre. This winning combination led to the exploration and characterization of
photobioreactors for microalgae production; the identification of novel bioactive microalgae
extracts and proof of concept of their use as nutraceuticals in aquaculture and
cosmeceuticals. The progress made was possible due to scientific and technological
advances that will be summarised in this Algae4a-b analytical handbook that carries on from
where the Algaecom handbook left off.
The Algae4a-b was set-up by a group of European scientists and producers convinced that
microalgae have the potential to revolutionize biotechnology and produce novel products
with high added value for nutrition, aquaculture and cosmeceuticals. Although microalgae
have been commercially cultivated for over 50 years, in depth molecular characterization is
necessary if they are to achieve their full market potential. The biological and chemical
diversity of the microalgae has been the source of unique bioactive molecules with the
potential for industrial development as pharmaceuticals, cosmetics, nutritional supplements,
molecular probes, enzymes, fine chemicals, and agrichemicals. Of the thirty thousand
microalgal species believed to exist, only a few thousand strains are kept in collections
around the world, only a few hundred have been investigated for chemical content. Because
they are largely unexplored, the microalgae biomass represents a rich source for discovery
in both academic and industrial sectors. The main scope of the Algae4a-b project is to
extend knowledge about microalgae and unmask their potential for industry focussing on
cosmetics and aquaculture. For this purpose, an integrated approach was pursued that
brings together: Microalgae and biomass production, use of -omics for high-throughput
analysis and innovation in aquaculture, nutraceuticals and cosmeceuticals
Microalgae and biomass production. The most cost-effective way to farm microalgae is in
large circulating ponds; however, a serious weakness in the use of outdoor ponds is the risk
of contamination by other microbes or indigenous algal species (Chen et al., 2011).
Alternative biomass production technologies such as closed photobioreactors provide
sterility and allow for much greater control over culture parameters such as light intensity,
carbon dioxide, nutrient levels, and temperature (Perez-Garcia et al., 2010). Selection of a
suitable production system clearly depends on the purpose of the production facility and
scope. One objective of the Algae4a-b project was to optimise photobioreactor systems for
microalgae cultivation with a view to establishing functional approaches to serve
cosmeceuticals and aquaculture demands.
Boosting of -omics for high-throughput analysis. Genomic technologies have spread in
the scientific community during the last decade due to the decrease of sequencing costs.
This field has rapidly expanded in the aquaculture sector and -omic resources are providing
useful tools for innovation and to improve competitiveness of the production sector (Cerda
and Manchado, 2013; Benzekri et al., 2014). In Southern Europe, the main aquaculture
finfish species are the gilthead seabream (Sparus aurata) and seabass (Dicentrarchus
labrax) although recently the Senegalese sole, Solea senegalensis, a high value species
worldwide has been adopted due to high market demand and collapse of the wild fishery. In
the context of the exploitation for aquaculture the basic biology and lifecycle and production
of these species is relatively well established. Moreover, next generation sequencing (NGS)
technologies have been used to generate transcriptomes in all three Mediterranean species

–6–
(Benzekri et al., 2014; Garcia de la Serrana et al., 2012; Sarropoulou et al., 2012; Tine et al.,
2014) and such resources constitute an important source of potential molecular markers,
including SSRs (simple sequence repeats, also known as microsatellites) and SNPs (single
nucleotide polymorphisms) and make implementation of complimentary techniques such as
microarray hybridization or qPCR and diagnostics possible. The available genomic
resources were used in the Algae4a-b to facilitate the evaluation on larval performance,
metabolism, health and welfare of microalgae nutraceuticals.
Innovation in Aquaculture. Aquaculture represents a relatively young industry that is
growing rapidly worldwide. The growth in the World population is projected to surpass 9
billion by 2050 and a major challenge is to ensure food security. The limitations of
terrestrial production mean that aquatic production systems are seen as a solution to this
problem particularly since marine species bring health benefits as they are low in saturated
fats and high in ω-3 fatty acids, vitamin D, iodine, selenium and other micronutrients.
Nowadays, fish provide ∼16% of the animal protein consumed by the world’s population and
with an annual global growth of 5% in aquaculture production this percentage is projected to
rapidly increase (Bostock et al., 2010). In Europe over 36 million people are employed
directly through fishing and aquaculture and as many as 200 million people derive direct and
indirect income from fish. Europe has changed from a World leader in aquaculture
production in the 1990’s to a net importer of fish and global production is currently dominated
by Asia with 89% of production volume and 79% of the value (Bostock et al., 2010). In the
context of the “Blue Growth Strategy” the EU Commission has recognized the contribution
aquaculture can make to boost growth and jobs in EU coastal and inland areas.
Innovation in Nutraceuticals. Nutraceuticals are bioactive compounds that when
incorporated in the diet can be used for the prevention or treatment of diseases. Many
substances that become bioavailable after digestion offer extra-nutritional beneficial
properties; these substances are called “bioactive”. The properties of such compounds
include antioxidant, anti-inflammatory, antimicrobial, anti- hypertensive, and anticancer
functions as well as regulatory roles for intra- and extracellular signalling pathways. The
nutraceutical market has grown in the last decade due to increased consumer knowledge
about these compounds and for their relevance in the prevention and therapy of several
diseases. In this context, in 2007, the nutraceutical market reached $117.3 billion, and it is
projected to grow to $176.7 in 2013 (Ranadheera and Vidanarachchi, 2013). In the USA, the
market was worth $241 billion in 2019 and is expected to increase to $373 billion in 2025.
Marine algae (macro and micro) have been identified as a most valuable source of bioactive
compounds with potential applications in the nutraceutical industry. These marine organisms
produce specific and potent active molecules that enable them to survive the characteristics
of the marine environment (Ranadheera and Vidanarachchi, 2013). Marine-derived
functional ingredients including certain enzymes, polysaccharides, methyl donors,
nucleotides, bioactive peptides, long chain omega-3 polyunsaturated fatty acids, vitamins,
minerals such as iron & iodine and carotenoids may possess anticarcinogenic, anti-
inflammatory, antihypertensive, antioxidant, hypocholesteroleic, anticoagulant, antidiabetic,
antimicrobial, immunostimulatory, appetite suppression and prebiotic properties enabling
them to be used as a part of nutraceuticals and functional foods.
Innovation in Cosmeceuticals. Cosmeceuticals are cosmetic products having drug-like
benefits that enhance or protect the appearance or order of the human body, which include
anti-aging creams, moisturizers, UV radiation protectors and skin whiteners. Cosmeceuticals
contain active ingredients such as vitamins, phytochemicals, enzymes, antioxidants and
essential oils and can be applied to products such as creams, lotions, and ointments (Kim et
al., 2008). An upcoming trend is multifunctional cosmetics, and microalgal extracts can

–7–
have this role as they can enhance both the appearance and the health status of the skin. In
cosmetics, algae extracts are used as thickening agents, water-binding agents, antioxidants,
to prevent blemishes, repair damaged skin, help seborrhea and inhibit inflammation (Kim et
al., 2008). Cosmetics companies claim the benefits of microalgae come from contents like
carrageenan, other algal polysaccharides, algal proteins or lipids, vitamin A, vitamin B1, iron,
phosphorus, sodium, copper, magnesium, calcium. It has been proposed that the antioxidant
properties of microalgae protect collagen and hyaluronic acid from breakdown and in this
way have anti-aging properties. There is considerable scope and opportunities for the use of
microalgae as cosmeceuticals.
Interesting molecules for exploitation in cosmeceuticals are polysaccharides. In contrast,
with terrestrial and marine plants, the structure and biological properties of microalgae
polysaccharides are mostly unexplored. The occurrence of polysaccharides based on
carbohydrate composition has been reported in many microalgae highlighting the wide
structural diversity of polysaccharides but only few data on fine chemical structure are
available (Hoagland et al. 1993). Biological assays conducted with microalgal
polysaccharide extracts have revealed some potent biological activity such as antiviral (Chen
et al., 2010), antioxidant, immuno-stimulating and reveals the diversity and potential of
microalgae (Yim et al., 2005; Suarez et al., 2005). Interestingly, the preliminary analysis of
Chlorella vulgaris has revealed the occurrence of large amount of rhamnose (Ogawa et al.,
1999) which usually present some appreciated texturizing effects in cosmetic formulation.
Chlorella pyrenoidosa contains an unusual arabino-galactan highlighting the diversity of
polysaccharides unrelated to green macro-algae or terrestrial plants (Suarez et al., 2005). In
Algae4a-b the functional properties of microalgal extracts, including the polysaccharides
were explored with a view to developing novel bioactive cosmeceuticals and also as
nutraceuticals for aquaculture.
Support of animal welfare and the use of cell-based animal models is a priority for research
and industry. Animal testing has been the method of choice for dermatotoxicological testing
for cosmeceuticals for decades. Decreasing acceptance of animal use in cosmetic testing in
the public arising from ethical objections forces the revision of traditional procedures and the
establishment of alternative methods. In addition, for certain endpoints including skin
irritation and genotoxicity, animal testing for cosmetics is already prohibited by the 7th
Amendment to the EU Cosmetics Directive. It is also important to realize that animals differ
from humans with regards to isoform composition, expression and catalytic activities of drug-
metabolizing enzymes (Martignoni et al., 2006). There is the urgent need for sophisticated
and well-characterized in vitro models as alternatives to animal testing to assess hazards for
human health with the overall goal of consumer protection. Various type of alternative
methods are being used for the assessment of potential dermal corrosion such as skin
models (Episkin, Epiderm)(Lawrence et al., 1997) and irritation such as in vitro primary
monolayer cultures, skin slices and reconstructed human skin equivalents (Basketter et al.,
1994). The in vitro tests incorporate numerous endpoints including cell morphology, viability,
membrane damage, metabolic effects and the release of various inflammatory mediators
(Botham, 2004). Algae4a-b developed and implemented a range of in vitro cell based
assays, including the development of an in vitro fish cell model for dermatological tests with
an aim to extending the scope of in vitro testing platforms for testing microalgae
biomolecules for cosmeceutical applications.
The present handbook presents methods and concepts arising from the progress made
during the 4 years of the Algae4a-b project (2016 – 2019). Most of the methods reported in
this handbook can also be found published in the context of scientific articles published by
project partners to ensure timely, public communication of progress. All the authors and

–8–
collaborators in this project appreciated the MSCA RISE scheme and the exciting
opportunities and stimulating environment the project offered. The opportunity to be involved
first-hand in translational research was a challenge but the outcome very fulfilling. We hope
you will find this analytical handbook useful for your own work and we would like to offer
thanks and acknowledge the essential role of the funding from Europe via H2020 MSCA-
RISE project 691102 (Algae4a-b).

Chapter 1. Microalgal culture

1.1 Background knowledge
Microalgae are unicellular organisms that can be found both in freshwater and marine water
environments and also on the surfaces of soils and rocks. They exhibit a high evolutionary diversity
which is easily observed by looking at the pigments used for the photosynthetic activity, or at the cell
wall structure and composition. Thus, microalgae can be found in a wide range of phylogenetic
groups in the eukaryotic tree of life (Tirichine and Bowler, 2011). A common feature to all of them is
the fact that growth rates are strongly affected by a combination of experimental parameters such as
light intensity, temperature, photoperiod, and nutrient composition of the media (George et al., 2014).
In that sense, a range of different culture media have been developed and used for both isolation and
cultivation of freshwater and marine microalgae. For each species, various permutations or even new
attempts might be necessary to establish the most suitable media for optimized growth and to
enhance production of bioactive compounds of interest and this may be inspired by habitat chemistry
or specific nutrient requirements of the microalgae (Anderson, 2005; Chew et al., 2018). Light quality
is a tool to stimulate photosynthetic activity and metabolic adaptation and coupled to optimization of
medium composition can improve not only growth but also pigment and biochemical composition of
the microalgae. In this regard, variations of medium composition and illumination have been exploited
to increase production of valuable products of different nature like lipids, carbohydrates, proteins and
pigments. And moreover, different media exhibiting different nutrient quantities can significantly
change production of cell biomass, which highlights that it is quite important to optimize appropriate
medium composition (Mandalam and Palsson, 1998; Bayona and Garces, 2014).
Microalgae are capable of absorbing light with optimal growth being observed with irradiance around
150 µmol/m2/s. However, when exposed to higher irradiance, they still can use the photon energy for
cell growth but photoinhibition is reported, which can also be caused by low irradiance (Sing and Sing,
2015). When dealing with outdoor cultivation this represents a major concern because of the natural
photoperiod and weather variability, reaching peaks even over 1000 µmol/m2/s in sunny days. Indoor
cultivation under artificial illumination has been proposed as a solution to outdoor cultivation instability
caused by unpredictable weather conditions, and hence fluorescent lamps, LEDs or photovoltaic cells
in the range of photosynthetically active region (PAR) are being used to create a platform for effective
microalgal production (Falkowski et al., 2004; Schulze et al., 2016). In this scenario, it should be
considered that both cell growth and metabolism are affected by light quality. Many microalgae can
exploit blue and red photons more efficiently thanks to pigments present in the light harvesting
complexes, and thus different light sources might be expected to modify the accumulation of desirable
cell products. For instance, red light photons have shown to accelerate cell cycle (Schulze et al.,
2016), while blue light has been reported to stimulate growth thus increasing biomass productivity and
chlorophyll a and accessory pigments (Valdiveloo et al., 2015; Ra et al., 2016). Green light has been
shown to have a positive effect regarding lipid production (Das et al., 2011; Ra et al., 2016). Light
intensity and photoperiods can also exert a significant influence on lipid and fatty acid content and
also pigment composition (Liang et al., 2001; Wahidin et al., 2013; Guermazi et al., 2014). Thus,
manipulation of light quality is a useful tool to manipulate microalgae growth and metabolism with
potential industrial applications bearing in mind that this will most likely be species-specific.
In the framework of the project Algae4A-B, the green microalgae Tetraselmis chuii (T. chuii) was
selected as the target species to study the influence of different media and light conditions on
microalgae growth as they are routinely cultured at Fitoplancton Marino, S.L. (FITMAR) premises.
Despite the previous extensive use of T. chuii in aquaculture, a clear protocol or methodology

–9–
describing the optical culture conditions for this species is lacking in a literature search. In this regard,
commercial laboratories usually employ the enriched medium f/2 (Guillard and Ryther, 1962) to
maintain strains, while others use 2-F medium. The effect of these two media has been previously
explored in T. chuii under different experimental variables (initial cell density and inoculation time),
and an interaction between initial inoculum density and the culture medium was observed (López-
Elías et al., 2011). The impact of different concentrations of heavy metals (zinc and copper) on growth
parameters has been also studied in T. chuii. Low concentrations of both metals (in the range of 5-10
mg/l) elicited a decrease in microalgae growth, but higher concentrations provoked an increase in the
culture period (Taha et al., 2012).
The development of lab-scale facilities for microalgae culture facilitates the investigation of the effect
of different culture parameters under controlled conditions and avoids uncontrollable variation typical
of outdoor conditions. In indoor conditions, it has been shown that light intensity and photoperiod
affect the growth and also nutrient uptake of T. chuii (strain PLY429). Under three different light
intensities (220, 110, and 73 µmol/m2/s), the best results regarding growth and nitrogen and
phosphorus uptake were obtained with the highest intensity and the longest light:dark cycle (24:0),
reaching values of division rate around 0.7 d-1 (Meseck et al., 2005). As expected, such results
highlighted that light limitation is a critical negative factor for the species growth. More research can
be found in the literature not specifically for T. chuii but for other species belonging to Tetraselmis
genus. For instance, in Tetraselmis sp. (strain V2), the combined effect of salinity, pH and irradiance
has been evaluated in terms of production of chlorophyll, carotenoids and starch, with high irradiance
(182 µmol/m2/s) being correlated with high starch and carotenoid content (Dammak et al., 2018). For
Tetraselmis suecica, a very close phylogenetically related species to T. chuii, a clear effect of light
intensity combined with nitrate concentration has been found for biomass production and oil
accumulation (Go et al., 2012).
The following methodologies outline the experimental protocols that can be used to study a range of
parameters that influence microalgae growth and the production of metabolites of interest.

1.2 Experimental protocol for selecting culture medium

-Prepare triplicate 250 ml flasks for each media to be tested (clean and sterile) and place 150 ml in
each (Figure 1.1). A stock culture of T. chuii (or any other microalgae strain) at the exponential
growth phase, cultured at controlled indoor conditions in medium routinely employed for microalgae
growth should be used for inoculation. Cell density of the stock culture should be determined using a
Neubauer chamber (~4 x 106 cells/ml), and then the necessary volume to reach an initial cell density
of 20000 cells/ml in each flask should be added.
-Before adding the microalgae to the flasks, the cells should be washed twice in sterile seawater.

Figure 1.1 Set-up of the experimental assay for the different growth media (day 0)

-The cultures should be maintained for 14 days at 23 ºC, with a light intensity of 150 µmol/m2/s, and
5% CO2 in bubbling air.
-Control the volume of culture daily by adding enough sterilized distilled water to reach the original
volume of 150 ml. Volume loss occurs due to evaporation.
-Remove an aliquot (around 5 ml) to determine the optical density at 678 nm (the peak of chlorophyll
absorbance in the case of T. chuii cultures, but this can be modified according to the microalgae
strain being evaluated) and at 750 nm, and count cells to monitor growth.
-Differences in growth are clearly observed by comparing the colour exhibited by the culture in the
flasks (Figure 1.2),

–10–
Figure 1.2 Cultures after 14 days of growth in the different media – note the difference in the colour of the media

Growth rates (µ) can be determined after the first 7 days of culture and at the end of the assay at 14
days using the following equation:
µ (divisions/day) = (LnBn – LnB0)/(tn – t0)
where B0 is the microalgae density at the beginning of the assay, Bn the microalgae density at 7 or 14
days (average of the three replicates), and (tn – t0) is the period (in days) of the culture since
inoculation (7 or 14, respectively).
Generation time can also be determined at 7 days and 14 days after inoculation using the following
formula (Kawaroe et al., 2015):
Tt (days) = (1/µ) x (tn – t0)
where µ is the growth rate, and (tn – t0) is the period (in days) of the culture since inoculation (7 or 14,
respectively).

1.3 Measuring the optical density (OD) of cultures

Optical density is used to monitor the growth of microalgae. In the case of the microalgae, T. chuii,
678 nm corresponds to the peak of chlorophyll absorbance. A second OD, 750 nm can be chosen to
assess the turbidity of the medium, which will change with cell growth but is not affected by
chlorophyll absorbance. The evolution of the OD of T. chuii microalgal cultures at 750 nm is similar to
that at 678 nm indicating both are useful to assess the change in biomass in cultures.

1.4 Experimental protocol for selecting the wavelength for microalgae culture
Different wavelengths can be obtained using color filters: red, blue and green (no filters are employed
as the Control). Absorbance and transmittance profiles should be determined for each light filter (see
Figure 1.3). Light intensity should be measured using a radiometer and adjusted to the desired value.

Figure 1.3 Transmitted and absorbed light by filters within the PAR region

–11–
-Use triplicate 1 L flasks as outlined above for each experimental condition.
-Put 625 ml of sterile, filtered seawater in each flask and then inoculate with a stock culture of the
microalgae of interest (we used T. chuii) in the exponential growth phase grown under controlled
indoor conditions in the medium routinely employed for microalgae growth.
-Cell density of the stock culture should be determined using a Neubauer chamber, and then the
necessary volume of inoculum should be added to each flask, so it reaches an initial cell density of
~770,000 cells/ml (day 1).
-Thereafter, the cultures should be maintained in the optimal growth medium for 5 additional days at
23 ºC, with 5% CO2 in bubbling air.
-Control the volume of culture daily to avoid losses caused by evaporation and add sufficient sterilized
distilled water to reach the original volume of 625 ml.

1.5 Evaluating microalgae growth and metabolism under different light

wavelengths
-Remove an aliquot (5–10 ml) of the microalgae from each mass to monitor growth at two days (day
3) and five days (day 6) after inoculation by progression of cell density using a Neubauer chamber.
-At the end of the trial (day 6), collect the biomass so it can be processed and analyzed for
metabolomic and gene expression analyses. Biomass should be collected by centrifugation (4,000
rpm, 4oC) and the resulting pellet immediately frozen in liquid nitrogen and freeze-dried.
-As an example, microalgae growth, in this case T. chuii, under different wavelengths is shown in
Figure 1.4. Although at day 3 the biomass (cell number) is very similar, a clear negative effect on
growth is observed at day 6 because of the modified wavelength of light the microalgae were
exposed to. The negative effect was most evident for the blue and green filters. The growth
inhibitory effect was less pronounced with the red filters compared to the control.

Cell density under different light filters

3,5
3,0
Million cells/ml

2,5

2,0

1,5
1,0

0,5

0,0
Red Blue Green Control
Light filters

Day 1 Day 3 Day 6

Figure 1.4 Evolution of T. chuii cell density with light filtering. The average of three flasks is represented + the standard error.

1.6 Determining the effects of red, blue and green light filtering on the
microalgae metabolism
-Grind freeze dried microalgae biomass (T. chuii) in a pestle and mortar and extract with 380 μl
methanol and 20 μl ribitol in methanol (0.2 mg/ml).

–12–
-Incubate at 70 °C for 15 min with continuous shaking and then add 200 μl of chloroform and incubate
for a further 5 min at 37 °C under continuous shaking.
-Add 400 μl of double-distilled H2O, vortex vigorously and then centrifuge at 18,000 g for 5 min at
room temperature.
-Transfer the aqueous phase containing the polar metabolite fraction to a new Eppendorf tube and dry
with a current of nitrogen gas. For derivatisation, dried samples should be re-suspended in 25 µl
methoxyamine-HCl (MOX) (20 mg/ml in pyridine) and incubated at 30°C for 90 min with continuous
gentle agitation.
-Add 50 µl of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) and incubate at 37°C for 30 min
with continuous gentle agitation. Finally add 10 µl of n-alkane mix for determination of retention
indexes (RIs).
-Gas-Chromatography coupled to Mass-Spectrometry (GC-MS) measurements can be performed with
an Agilent Technologies 7890A GC system coupled to a Agilent Technologies 5975C MS (Agilent
Technologies, Frankfurt, Germany (or similar equipment).
-Obtain metabolome data of microalgae biomass under the different light conditions in order to
correlate metabolomic content with the respective transcriptomic profiles. The metabolome results
can be expressed as a response that corresponds to the ratio between the areas of the target
metabolite divided by the area of the reference metabolite (ribitol, m/z 319) and reported relative to
the dry weight.

1.7 Determining the effects of red, blue and green light filtering on the
microalgae transcriptome
-Isolate high-quality RNA samples from cell biomass collected at the end of the trial using a Qiagen
total RNA extraction kit and following the manufacturer’s instructions (see section 5).
-Process total RNA for library construction, amplification and sequencing using an Illumina TruSeq
RNA Sample Preparation Kit v2.
-Construction of the sequencing library involves purification of the poly-A containing mRNA molecules
using oligo-dT attached magnetic beads.
-Fragment the isolated mRNA into small pieces using divalent cations under elevated temperature.
The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and
random primers and then the second strand cDNA is synthesized using DNA polymerase and
RNase H.
-After, the cDNA fragments go through an end repair process, addition of a single ‘A’ base, and
ligation of the adapters.
-Finally, the reaction products are purified and enriched with PCR to create the final cDNA
sequencing library.
-RNA-sequencing can be done with an Illumina MiSeq. Raw reads obtained should be filtered and
trimmed to remove adapters, primers and low-quality reads.

1.8 Establishing experimental facilities for indoor trials

Lab-scale facilities for microalgae growth can
be readily set-up. An example of facilities set-
up in the context of Algae4a-b is provided in
this chapter. The facilities consist of a set of 1 L
and 2 L photobioreactors in which the behavior
of different microalgae species can be tested
under highly controlled growth conditions. This
allows fast screening and the identification of
the best parameters for microalgae growth (e.g.
biomass productivity), metabolite and bioactive

Figure 1.5 Pilot scale experimental facility for indoor assays –13–
to increase the throughput of condition screening.
compound production. Parameters that can be regulated in the system include pH and CO2
concentration and light intensity and temperature (see Figure 1.5). The system can be built of
modules, which allows easy addition of additional facilities for multiplexing to assess a greater number
of parameters or species/ strains simultaneously.
-Similar facilities as described above can be used to assess the effect of different temperatures (e.g.
20 and 25 ºC) or light intensities (e.g. 50 and 150 µmol/m2/s) on T. chuii growth. If a control system
for pH is available, it can also be controlled and the effect of pH (e.g. pH 7 and pH 8) can be tested.
-Inoculate T. chuii in 650 ml of f/2 medium using the same cell density as previously described.
-Grow microalgae cultures using variable conditions and take samples periodically over 2 weeks.
Determine cell number at each sampling point using a Neubauer chamber and measure nutrient
consumption by UV spectrophotometry.
-As described in other experiments the volume in the flasks has to be maintained constant by the
addition of sterile double distilled water daily.
-Construct growth and nutrient consumption curves. An example of expected results is provided
below (effect of light intensity in Figure 1.6 and nitrate and phosphate consumption of T. chuii in
Figure 1.7):

Figure 1.6 Combined effect of light intensity, temperature and pH on T. chuii growth

-The pilot system set-up allows several conditions to be simultaneously tested, thus evaluating the
combined effect of light intensity, temperature and pH on T. chuii growth or other microalgae
species.

2
Figure 1.7 Nitrate and phosphate consumption of T. chuii cultures at 25 ºC and 150 µmol/m /s

Monitoring of the use of nutrien

Tetraselmis chuii: nitrate consumption at 25 ºC, pH 7 Tetraselmis chuii: phosphate consumption at 25 ºC, pH 7
4500 250
4000
Pho sph ate co n cen tratio n (µM)

200
Nitrate co ncen tratio n (µM )

3500
3000
150
2500
2000
100
1500
1000 50
500
0 0
0 1 4 8 12 15 0 1 4 8 12 15
Time (days) Time (days)
ts is
also possible using the pilot-scale indoor bioreactor and nitrate and phosphate consumption can be
established by spectrophotometry.
-The pilot system can be used for screening a range of experimental conditions and growth and to
determine metabolite production. The challenging step is to transfer from the pilot scale to the
industrial scale bioreactor.

–14–
–15–
Chapter 2. Transfer of the optimal growth conditions from lab scale
to large scale photobioreactors
2.1 Background knowledge
Microalgae have been investigated as a promising renewable feedstock for a range of very different
applications, including fuel or chemical production, as a source of bioactive compounds with health
benefits for the food or cosmetics industry, or in the aquaculture and aquariology sector. Compared to
land plants, microalgae offer several advantages including potential high yields or utilization of non-
arable lands. But drawbacks remain, due to the cost and efficiency of the culture processes and
strong barriers to major development of industrial processes based on microalgae production. Despite
their enormous potential it is considered that the industrial use of microalgae is still at an early stage
of development. Optimization of design, operation and control of photoproduction processes have yet
to be investigated in depth, in particular in the context of scaling-up the production capacity to
increase the portfolio of microalgae-based processes towards different applications (Draaisma et al.,
2013).
In a real-life scenario, culture conditions at an industrial scale differ drastically from the optimal
standard conditions usually employed at a laboratory scale for microalgae growth and hence scaling
up from laboratory to production is linked to significant productivity losses. Thus, optimization and
control of microalgae growth in outdoor conditions is required but is challenging as the key state
variables of the cultivation system such as temperature or light availability continuously vary due to
fluctuating meteorological conditions as well as the day-night cycles. This is a critical point, and so
different models have been developed to predict microalgae growth from expected weather conditions
(De-Luca et al., 2018). In any case, biomass productivity is largely dependent on a wider range of
cultivation parameters including, in addition to temperature and light, the microalgae strain, nutrients,
pH, mixing, and purity of the culture (Rizwan et al., 2018). Hence, these variable factors should be
controlled when possible with reliable technology-based systems in order to ensure biomass
production with similar features between production cycles.
In the setting of Algae4a-b, specific and optimized parameters set-up at Fitoplancton Marino, S.L.
(FITMAR) premises for microalgae growth in large-scale photobioreactors were established in a
Report of methods for the industrial scale production of controlled quality biomass, which will be
briefly described. First the basic methodology used for scaling up production and monitoring the
metabolome of the microalgae target species T. chuii will be described when cultured under outdoor
variable conditions and then control of biomass quality will be considered. The key parameters
recorded in real-time are reported and to assess the variability of production replicate bioreactors
were analysed to assess differences that may exist between samples collected at different times of
the day owing to changes in the non-controlled parameters temperature and light intensity due to the
natural photoperiod.

2.2 Production at industrial scale of T. chuii in outdoor conditions

To establish the conditions for scale-up from the laboratory to outdoor industrial scale production,
experimental analysis was carried out to establish how parameters identified in controlled in door
cultures changed. The approach is presented below to provide a working protocol of the approach
and factors that can be used to monitor the outcome of scale-up.
To ensure clonal cultures, scale-up should begin from a single isolated colony of the microalgae
target species.
This can be achieved using one of the following strategies:
1) Cell dilution technique, in which an aliquot is removed from the microalgae culture, and serial
dilutions are performed and dispensed into multi-well plates with culture medium. In each cycle the
original sample is diluted increasing the probability of single-cell isolation, and stops when it is
probable that no cell will be transferred.

–16–
2) Streak plating technique, which allows a single colony to be selected from agar plates. In this case,
a loop is loaded with microalgae culture, and then this sample is spread with the loop across the
agar. Thereafter, the agar plate is incubated under controlled conditions until colonies appear.
The first technique is most often used with field samples with the goal of discovering new species,
whereas the second one is more recommended for routinely cultured species (Figure 2.1), although it
should be considered that not all microalgae species can grow on agar.

Figure 2.1 Overview of the up-scale process for well-established microalgae, starting from an isolated colony and scaling-up
through progressively larger volumes to the outdoor production level (Fitoplancton Marino, S.L. facilities).

The industrial scale production of microalgae is divided into two stages:

1) Indoor controlled conditions,
-All steps up to SC2 (see below) are developed in an indoor controlled culture chamber (temperature:
17 ºC, irradiance: 150 µmol/m2/s), and thereafter in an indoor controlled culture room (temperature: 22
ºC, irradiance: 150 µmol/m2/s obtained using daylight fluorescent tubes).
-Briefly, an aliquot of a microalgal culture in growth medium f/2 that is in the exponential phase is
seeded onto a marine agar plate using a 10-µl loop. The streak-plate procedure is used to obtain
isolated colonies after 2-3-weeks under controlled growth conditions.
-An isolated colony is picked from the marine agar plate and inoculated into 2-3 ml f/2 medium in a 10
ml sterile culture tube. This inoculum is incubated for 2-3 weeks without shaking (Subculture-1;
SC1).
-SC1 is then inoculated into a 50 ml flask with 30 ml f/2 medium (~1/10 dilution), and incubated during
2-3 weeks without shaking (SC2).
-SC2 is used to inoculate a 1 L flask with 700 ml f/2 medium (~1/15 dilution) and 5% CO2 enriched air,
and then allowed for growing for 7 days (SC3).
-SC3 (~400 ml) is inoculated into a 5 L balloon with 4 L f/2 medium (~1/10 dilution) and 5% CO2
enriched air and allowed for growing for 7 days (SC4).
-Finally, the SC4 is inoculated into a 50 L photobioreactor with f/2 medium (~1/10 dilution) and 5%
CO2 enriched air and allowed for growing for 7 days (SC5).
2) Outdoor conditions.
Depending on the species of microalgae a different concentration of biomass is used. In the case of
Tetraselmis chuii 1/3 dilution is employed to inoculate a single outdoor photobioreactor with f/2
medium and pure CO2 under automatically controlled temperature and pH conditions.
Growth of the microalgae is allowed to proceed for 2-3 weeks so that the culture is in the log phase
before the start of the semi-continuous phase.

–17–
3) Semi-continuous outdoor phase.
At this stage, and depending on the season, cultures can be maintained from 2 to 4 months. Daily,
10% to 20% of the volume of the culture is harvested and replaced by the same volume of f/2
medium. A daily control of nutrient consumption is performed to ensure no deficiency of nutrients
during the culture.

2.3 Determining growth performance of indoor and outdoor industrial

bioreactors and identifying factors influencing microalgae growth
Comparative analysis of growth performance can be carried out between indoor (5 L balloons) and
outdoor (photobioreactor) cultures of the species T. chuii.
The procedures outlined above for the set-up of indoor cultures can be also applied here. In the case
of indoor cultures, controlled conditions can be used (e.g. light intensity: 150 µmol/m2/s, temperature:
22 ºC) and growth allowed to proceed until the desired cell density is reached (~1.8 x 106 cells/ml at
day 11 for batch culture in the example shown in Figure 2.2).
In the case of the outdoor photobioreactor a similar starting microalgae biomass can be used for
cultures and the medium is the standard f/2. Culture conditions vary with environmental conditions
and the final microalgae density is recorded after 18 days in semi-continuous cultures (Figure 2.2
gives an example, values collected in March-April 2017).

Cell density NO3 consumption Cell density

2 2000 12

1,6 1600 10
Million cells/ml
Million cells/ml

8
1,2 1200
µM NO3

6
0,8 800 Beginning of semi-continuous phase
4

0,4 400 2

0 0 0
0 1 3 4 5 6 7 9 11 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Days of culture Days o f culture
March-April 2017

Figure 2.2 Comparative growth of T. chuii cultures under controlled indoor (left hand side) and variable outdoor conditions
(right hand graph)

The results of the growth experiments under controlled indoor conditions suggests that the growth-
limiting factor is light availability when compared with the microalgae densities achieved in outdoor
conditions.

2.4 Determining the effect of light intensity and temperature on T. chuii

biomass under outdoor conditions
T. chuii is an ideal experimental model to study scale-up parameters as: i) the species can be grown
all around the year and does not exhibit temperature sensitivity, ii) from a scientific point of view, it is a
much less studied species when dealing with -omics (transcriptomics or metabolomics), and iii) given
the high antioxidant activity shown by this species, the results have high potential interest for cosmetic
and aquaculture applications.
To assess the impact of light on growth of T. chuii:
-Prepare 3 outdoor photobioreactors (as outlined above).
-Sample the photobioreactors during three consecutive days, both early in the morning (just before
dawn) and at midday (with maximum light intensity), for -omics analyses. Several litres of the
microalgae culture is needed.
-Centrifuge the samples (4,000 rpm, 4ºC) and then immediately freeze the resulting pellets in liquid

–18–
 nitrogen, and there after freeze-dry.
-Collect radiation data using a radiometer early in the morning and at midday (Figure 2.3).
Daily irradiance (PAR)
1400

1200

1000

µmol/m2/s
800

600

400

200

Time

Samples collected for ‐omics + dry weight Samples collected for gene expression + dry weight

Figure 2.3. Light intensity values measured early in the morning and at midday (peak value) to assess the variability of light in
outdoor industrial scale photobioreactors during November 2017.

-Measure the temperature in photobioreactors using a temperature probe during the early morning
and at midday (Figure 2.4).

Figure 2.4 Temperature variation from dawn and midday during November 2017 in industrial scale outdoor photobioreactors.

The results of this simple analysis reveal that the two main parameters that affect biomass quality in
outdoor conditions are light irradiance and temperature and that they are extremely different at
different times of the days. This is relevant as it shows that controlled lab conditions cannot simulate
the real-life outdoor industrial scale production and that it is essential after rapid pilot scale screening
to test the performance of microalgae in a production scale bioreactor. The effects of factors such as
cell density or pH can be more easily controlled in the outdoor photobioreactors as they are computer
controlled. Temperature and light are the important and determinant factors for the non-comparability
of indoor and outdoor scale-up.
Having demonstrated the value of laboratory-based screening as a rapid high throughput method for
identifying conditions that may improve microalgae biomass productivity, it is clear that they have to
be validated in a production setting.

2.5 Experimental analysis of microalgae biological activities of interest

The influence of outdoor production conditions on activities of interest in microalgae biomass can be
assessed taking into consideration the daily cycle. This allows the identification of the optimal time of
harvest of the microalgae biomass. Typical activities and assays that can be used include the
antioxidant capacity, Superoxide dismutase and transcriptome analysis. A detailed description of the
assays is given in the Algaecom handbook and so only a cursory presentation is provided below.
2.5.1 Antioxidant capacity of T. chuii biomass harvested at different times of the day

–19–
Total antioxidant capacity of extracts obtained from microalgae biomass collected at dawn and
midday is analyzed using the Ferric ion Reducing Antioxidant Power (FRAP), an antioxidant capacity
assay that uses Trolox as a standard (Benzie and Strain, 1996). The method is based on the
formation of an O-Phenanthroline-Fe(2+) complex and its disruption in the presence of chelating
agents (such as EDTA). The principle steps of this assay are described below:
-Prepare the following reagents: 1) sodium acetate buffer 300 mM, pH 3.6 (as an example, weigh 3.1
g of sodium acetate trihydrate and add 16 ml of glacial acetic acid, then add water to reach 1 L final
volume), 2) TPTZ (2,4,6-tripyridyl-s-trianzine) 10 mM in 40 mM HCl, and 3) FeCL36H2O 20 mM. The
working FRAP reagent is prepared by mixing 1+2+3 in a ratio 10:1:1 just before use.
-Prepare a stock of standard solution, for instance trolox or ascorbic acid. From this stock, prepare
serial dilutions for the calibration curve.
-Microalgae extract preparation:
 Weigh 100 mg of freeze-dried T. chuii and add to 1 ml of Milli-Q water in a 2 ml safe-lock
Eppendorf tube (10% biomass). Vortex for 30 s at maximum speed, and keep on ice for 30
min for hydration, repeat vortexing every 10 min.
 In parallel, prepare two 1.5 ml safe-lock Eppendorf tubes with two spoonful’s of stainless-steel
beads (SSB) 0.2 mm (~200-225 µl) and add 600 µl of Milli-Q water. Vortex for 10 s at
maximum speed.
 Then, add to each tube 200 µl of hydrated microalgae cell suspension (1/4 dilution; 25
mg/ml). Cell lysis is performed in the cell disruptor Bullet Blender 24 (Next Advance) at v=10
for 3 min.
 Add 8 µl of NaOH 10 N (0.1 N final concentration) and vortex for 10 s at maximum speed.
Incubate at room temperature for 2 h with continuous shaking (220 rpm), mixing the content
of the tubes every 30 min by inversion.
 Centrifuge at 13,000 rpm for 10 min and transfer 600 µl of the supernatant to a clean 1.5 ml
Eppendorf tube.
 Finally, centrifuge again at 13,000 rpm for 5 min, and transfer 500 µl of supernatant to a clean
1.5 ml Eppendorf tube, and keep on ice.
-The assay procedure includes then the following steps:
 Mix 100 µl of microalgae extract with 3 ml of working FRAP reagent, vortex, and measure
absorbance at 593 nm (time 0).
 Blank (Milli-Q water) and serial dilutions of the standard are processed in the same way.
 Heat all samples at 37 ºC for 4 min, and measure again at 593 nm.
 Determine the change in absorbance of the microalgae extract from 0 to 4 min.
 Construct a standard calibration curve by plotting the 593 nm values (y-axis) versus their
concentration (x-axis).
 Determine the FRAP value of the microalgae extract from the absorbance value using the
calibration curve. If the microalgae extract was diluted, adjust the final FRAP value by
multiplying by the employed dilution factor.

2.5.2 Superoxide Dismutase (SOD) capacity of T. chuii biomass

This assay relies on the reduction of the dye WST-1 (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)- 2H- tetrazolium, monosodium salt) with a superoxide anion (see Algaecom handbook
for full details).

Figure 2.5 Superoxide dismutase activity assay with cell-free extracts of T. chuii harvested across three days in November

–20–
2017.

The rate of the reduction with O2 is linearly related to the xanthine oxidase (XO) activity, and is in turn,
inhibited by SOD, as shown in Figure 2.6. Subsequently, the IC50 (50% inhibition activity of SOD or
SOD-like materials) can be determined as a decrease in the absorbance at 440 nm which is the
absorption maximum of WST-1 and is proportional to the amount of superoxide anion.
An example of SOD activity using freeze-dried biomass of microalgae (T. chuii) collected at dawn and
midday is shown in Figure 2.7 and no significant changes were detected.
Relative SOD activity Relative SOD activity
PB R14‐2 PB R14‐3 PB R14‐6 1,20
1,20 1,00
1,00

Arbitrary units
0,80
Arbitrary units

0,80
0,60
0,60
0,40
0,40
0,20 0,20

0,00 0,00
Morning Midday Morning Midd ay
Sampling time Sampling time

Figure 2.6 Relative SOD activity of T. chuii cell-free extracts

2.6 Metabolomic analysis of biomass collected early in the morning and

midday
2.6.1. Sample collection
For metabolomic analysis, collect T. chuii biomass from three outdoor photobioreactors during three
consecutive days, both early in the morning (before dawn; referred to as Morning) and at midday
(referred to as Midday). For each sample:
 Centrifuge 1 L of culture per photobioreactor in several steps (a total of five) using 4 x 50 ml
Falcon tubes, at 5,000 rpm for 5 min.
 Thereafter, wash the cell pellets twice with ammonium formate 0.53 M (all the microalgae
cells from the same photobioreactor are joined in a single tube).
 Store immediately each single cell pellet at -80 ºC, and finally freeze-dry and store at -20 ºC.
2.6.2 Analysis of the metabolome
-Grind 50 mg of lyophilized microalgal samples with liquid nitrogen. For each treatment, three
independent biological repeats can be analyzed, and two independent metabolite profiles obtained
for each biological repeat (n=6).
-Grind the pulverized biomass with 395 μl methanol and 5 μl of 1 mg/ml ribitol. Incubate samples at
70°C for 15 min under continuous shaking. Then, add 200 μl of chloroform and incubate at 37°C for
5 min under continuous shaking.
-Add 400 μl of ddH2O followed by vigorous vortex and centrifugation at 13,000 rpm for 5 min at room
temperature.
-For derivatization resuspend the dried samples in 50 µl of methoxyamine-HCl (15 mg/ml in pyridine)
and sonicate for 10 min at room temperature.
-Incubate at 50°C for 1 h and sonicate for another 10 min at room temperature.
-Rapidly add 50 µl of N-Methyl-N-(trimethylsilyl)-trifluoroacetamide to the sample with 1%
trimethylchlorosilane (MSTFA + 1% TMCS) and incubate at 50°C for 1 h.
-Inject a n-alkane mix separately, for the determination of retention indexes (RIs) by GC/MS.
-GC/MS analysis can be carried out using an Agilent Technologies 7890A GC system coupled to an
Agilent Technologies 5975C MS (Agilent Technologies, Frankfurt, Germany). Separation is achieved
on a DB-5MS fused capillary column (60 m long, 0.25 mm internal diameter, film thickness 0.25 μm,
J&W Scientific). Helium is used as the carrier gas at a flow rate of 1 ml/min. Split mode of injection is

–21–
 used and the injection volume is 1 μl. The GC inlet temperature is held at 280°C.
-Set the oven temperature program as follows: initial temperature 80°C (held for 2 min), followed by
an increase to 315°C (held for 12 min) with rate change of 5°C/min. Total run time is 60 min. The
MS source is held at 250°C and the quadruple at 150°C, scan from m/z 50-650.
-AMDIS (Automated Mass Spectral Deconvolution & Identification System) software, provided by
NIST (National Institute of Standards and Technology), is used to extract information about the
compounds analyzed by GC-MS. Compound libraries Feihnlab and Golm metabolite databases are
used for the identification of each component, according to its mass spectrum and retention time.
-Calculate the compound levels as the relative response ratio of peak areas of the target metabolite
related to the peak area of the reference metabolite (ribitol, m/z 319) and normalize with respect to
the sample dry weight.
-Use partial least squares discriminant analysis (PLS-DA) to analyse the metabolome data, using
Multibase software. Evaluate statistical significance of the results by analysis of variance (ANOVA)
followed by Tukey’s HSD (Honestly Significant Difference) or Fisher's Least Significant Difference
(LSD) multiple comparison tests at a 95% level of significance (p<0.05).

2.7 Industrial scale production of controlled quality biomass

In a real-life scenario, microalgae culture conditions at an industrial scale differ drastically from the
optimal standard conditions usually employed in the lab for microalgae growth and hence significant
productivity losses can be observed. Thus, optimization and control of microalgae growth in outdoor
conditions is quite complex because the key state variable of the cultivation system such as
temperature or light availability continuously vary due to fluctuating meteorological conditions as well
as the day-night cycles. This is a critical point, and so different models have been developed to
predict microalgae growth from expected weather conditions (De-Luca et al., 2018). In any case,
biomass productivity will be largely depending on a wider range of cultivation parameters including, in
addition to temperature and light, the microalgae strain, nutrients, pH, mixing, and purity of the culture
(Rizwan et al., 2018). Hence, these variable factors should be controlled (whenever it is possible) with
reliable technology-based systems in order to ensure reproducible biomass production from culture to
culture.

2.8 Up-scaling of microalgae cultures

A prototype and optimized method has been developed to increase biomass productivity in outdoor
photobioreactors (see chapter 2.2).
A computer-controlled system for key parameters regulating microalgae growth has been developed
at FITMAR for tubular photobioreactors. One production unit contains 6 photobioreactors, these
photobioreactors are composed of the phototube were microalgae perform photosynthesis and a
degasser tank were gas exchange is achieved. A computer controlled system monitors in real time
temperature and pH of each photobioreactor, as well as the state of the set of electronic valves
regulating the flux of water or CO2, switching on/off the cooling system, as well as daily culture
harvesting during the semi-continuous phase for biomass production.
These technological developments are critical to standardize and ensure the best and optimal quality
of the produced biomass using large-scale outdoor photobioreactors. This control system permits
different growth conditions for the different species to be set (either optimal or to enhance the
production of metabolites of interest) and permits production of consistent batches with the same
properties during different periods of the year.

–22–
Chapter 3. Purification and structural analysis of microalgae
polysaccharides
3.1 Background knowledge
Polysaccharides can be found in all organisms and exhibit a great variability of biochemical structures
and composition (Delattre et al., 2016). In the last years, these molecules have received much
attention owing to their wide range of biological activities, such as immunomodulatory, antibacterial,
anticoagulant, antimutagenic, radioprotective, anti-oxidative, anti-ulcer, anticancer, or anti-
inflammatory. The diversity of carbohydrate residues and the number of their possible linkages
explain the wide structural diversity of polysaccharides. The carbohydrate polymer backbone is often
decorated with organic (e.g. methyl, pyruvate, acetate) or inorganic derivatives (e.g. sulphate), further
increasing the complexity of these macromolecules. In the oceans, polysaccharides represent the
most abundant biomass produced by photosynthetic organisms, including uni- and multicellular
species: micro- and macroalgae, respectively. Structural analyses of marine polysaccharides have
primarily focused on the cell wall polysaccharides of macroalgae due to their economic importance.
Microalgae play a significant role in the ocean biogeochemical cycle; in particular, in terms of the
fixation of global carbon, they account for about 20% of total photosynthesis. These organisms have
attracted more and more attention and harbour economic value as sources of novel molecules, as
producers of lipids for bioenergy and as cell factories. Despite the recognized importance of these
organisms, the structure of microalgal polysaccharides has been virtually unexplored in contrast to
those of macroalgae. Composition analyses of microalgal polysaccharides located in the cell wall,
secreted or used for energy storage reflect the very broad diversity of polysaccharide structures.
The physiological functions of polysaccharides present in microorganisms are extremely diverse and
depend on their structure and architecture. They provide carbon and energy reserves, and are often
excreted (exopolysaccharides, EPS), particularly under physiological stress. Microalgae, and more
specifically cyanobacteria, can excrete large quantities of EPS, with potential biotechnological
applications in the food, pharmaceutical and cosmetic industry (Bhunia et al., 2018). However, the
molecular mechanism underlying EPS biosynthesis are yet poorly known (Schmid et al., 2015).
Moreover, universal methods for extraction and further purification are lacking, as these processes
largely depend on the origin and composition of the EPS. In this context we analysed the composition
of EPS isolated from 17 cyanobacteria strains and evaluated the impact of culture conditions. These
extracts were tested as potential bioactive in cosmeceutical formulations.
The storage polysaccharide of microalgae, chrysolaminarin, is made of a (1,3) glucan chain
decorated by branched (1,6) glucose. In the frame of the project, we discovered that
chrysolaminarin-enriched extracts had positive effects on the immune system of Senegalese sole
(Solea senegalensis) (Carballo et al., 2018a; 2019). The degree of polymerization as well as the
degree of branching depends on the biological origin of chrysolaminarin. Therefore, a protocol
allowing the extraction of the -glucan from an industrial valorisation perspective and to characterize
-glucan enriched fractions from the bacillariophyte Phaeodactylum tricornutum and the haptophyte
Tisochrysis lutea were implemented.

3.2 Selection of EPS producing cyanobacteria strains

-Centrifuge about 2 ml of a fresh culture of cyanobacteria and resuspend in
1 ml of deionized water.
-Put a drop of the cyanobacteria suspension on a glass slide and add a
drop of Indian ink and observe using an optical microscope.
-The black areas correspond to the part of the sample full of Indian ink and
clear area where no ink can diffuse is attributed to matrix likely containing
polysaccharides (Figure 3.1 shows a typical microalgal strain selected for

–23–
 further analysis).
-The optical microscopy observation is confirmed by the Dubois’s method (Dubois et al., 1956,
Montreuil et al., 1963), which is a colourimetric assay for determination of
Figure 3.1 Optical micrograph total sugar contents.
of cyanobacteria (green)
producing exopolysaccharide.

3.3 Analytical methodology

3.3.1 Purification of cyanobacteria EPS
-Depending on the microalgal strains, polysaccharides are deposited as a biofilm at the surface of the
incubator (or Erlenmeyer) or remain bound to the cyanobacteria cells.
-To isolate the EPS, cyanobacteria suspensions are precipitated in bleaching solution.
-The samples of cyanobacteria are initially green and should become clear yellow. The mix should be
centrifuged (5 min, 5,000 rpm at 4°C), the supernatant is discarded and the pellet is transferred to
water and dialyzed.
-After dialysis, the transparent solution may contain some insoluble matter (likely cell debris). The
particulate matter is eliminated by centrifugation (10 min, 5,000 rpm at 4°C).
-The polysaccharides occurring in the supernatant are precipitated by adding an excess of
isopropanol (1 vol sample/10 vol isopropanol). The polysaccharides should be evident as a solid
white precipitate.
-The polysaccharides are recovered from the sample/isopropanol mix by centrifugation (10 min, 5,000
rpm at 4°C).
3.3.2 Analysis of osidic composition
The molar ratio of monosaccharides can be determined from EPS samples using the per-O-
trimethylsilyl methyl glycoside method (Montreuil et al., 1986).
The protocol for sample processing is as follows:
-A total of 0.4 mg of sample and 50 μg of myoinositol (internal standard; Ino) is placed in a dry bath
with 500 μl of methanol/hydrochloric acid 3 N for 4 hours at 110°C. If it is not possible to weigh 400
μg of sample, a solution of sample at 1 g/l in water can be prepared and used. In this case, 400 μl of
the solution is freeze-dried in three tubes.
-After cooling at room temperature, samples are neutralized with silver carbonate, and then 50 μl of
acetic anhydride are added in order to reacetylate osamines potentially present.
-After one night in the dark and at room temperature, samples are centrifuged for 15 minutes at 3,000
rpm and supernatants are evaporated under a nitrogen jet.
-The compounds are dissolved in 70 μl of pyridine and incubated overnight at room temperature with
70 μl of Silyl reagent. After gentle evaporation of excess reactants under a nitrogen jet, the
trimethylsilylated methylglycosides are suspended in 700 μl of methylene chloride and analyzed by
Gas Chromatography (for instance, System GC-6850 AGILENT, in-column injection, FID
detector:flame ionization). The carrier gas used should be hydrogen. The HP-5MS column (30 m x
0.25 mm internal diameter) should be nonpolar. The temperature program is as follows: 120°C
maintained for 1 min and then a gradient of 1.5°C/min to 180°C followed by a gradient of 2°C/min to
200°C.
-Identify each monosaccharide by comparing its retention time relative to the internal standard with
those of pure standard monosaccharides treated under the same conditions. A response coefficient
can be calculated for each monosaccharide relative to the internal standard in order to define the
proportion of each monosaccharide within the polysaccharide analyzed. When possible, triplicate
samples should be analyzed.

–24–
An example is provided (Table 3.1 and 3.2) of the typical mass percentages of each monosaccharide
in 100 g of sample. The % hydrolyzed values represent the total mass of sugar (in g) in 100 g of
sample. And finally, the molar ratio represents the ratio of each monosaccharide compared to the
most abundant monosaccharide present in the sample. Monosaccharides are abbreviated in the
following way: Ara: arabinose, Fuc: fucose, Gal: galactose, GalA: galacturonic acid, Glc: glucose,
GlcA: glucuronic acid, Man: mannose, Rha: rhamnose, Rib: ribulose, Xyl: xylose.

Table 3.1 Sugar and protein content of 6 different production batches of microalgae

Sample Batch Species Fraction % Sugar % Protein

1A P. tricornutum Fraction 1 2,50 0,55

L310317
1B P. tricornutum Fraction 2 7,41 0,88
2A P. tricornutum Fraction 1 23,68 0,58
2B P. tricornutum Fraction 2 15,21 1,27
L310317
2C P. tricornutum Fraction 3 22,83 1,27
2BSol P. tricornutum Fraction 4 16,94 1,71
3A P. tricornutum Fraction 1 19,83 0,92
L100314
3B P. tricornutum Fraction 2 11,11 3,58
4A P. tricornutum Fraction 1 16,82 1,86
L310317
4B P. tricornutum Fraction 2 12,32 2,23
5A T. lutea Fraction 1 30,09 1,70
L300517
5B T. lutea Fraction 2 13,11 1,62
6A P. tricornutum Fraction 1 19,64 1,90
L100314
6B P. tricornutum Fraction 2 23,45 1,06

Data in Table 3.1 correspond to the analysis of a set of different -glucan enriched fractions obtained
from different microalgae production lots. Typical variation in the sugar content of the partially purified
sample is presented. In some fractions, percentage of sugars reached values of ~30%, but in most of
them values ranged between 11-25%. The reason for the variability is linked to the strain/species of
microalgae and other parameters such a time of sampling, duration of the production cycle and age of
culture, which probably influence the abundance of sugar. Based on the lab-based production and
scale-up analysis (chapter 2) the time of day that sampling is carried out has a big impact on a variety
of parameters. The protein content of the Table 3.2 Monosaccharide composition of 3 different microalgae samples
crude algal extracts is always much lower
than the sugar content and varies from 0.5–  ‐Glucan (CNRS)

3.58%. % mass * Sample 2A Sample 3A Sample 5A Extract P. tricornutum

(07/2018)
An example of the outcome of GC analysis of
Fuc 1.18 1.17 +/‐ 0.01 ‐ 0.33 +/‐ 0.03
the monosaccharide composition of the
sugar fraction of the microalgae is given in Ara ‐ ‐ 1.88 +/‐ 0.1

Table 3.2. An example of a -glucan fraction Gal 1.06 1.23 +/‐ 0.3 2.20 +/‐ 0.1

isolated from P. tricornutum (2018) is also Glc 2.08 0.63 +/‐ 0.1 12.06 +/‐ 0.6 21.86 +/‐ 3.6

included. This gives a clear example of the Xyl 0.91 1.07 +/‐ 0.1 1.37 +/‐ 0.1 1.15 +/‐ 0.2
variability of the monosaccharide Man 0.43 ‐ ‐
composition of the sugar fraction isolated Rha 1.01 1.30 +/‐ 0.2 ‐
from different samples of the same species GlcA ‐ ‐ ‐
of microalgae (Table 3.2). Except for sample GalA ‐ ‐ ‐
3A, Glc was the most abundant
Rib ‐ ‐ 2.00 +/‐ 0.1 6.62 +/‐ 0.8
monosaccharide, and together with Xyl these
% Total* 5.5 5.4 +/‐ 0.6 19.51 +/‐ 0.9 30.41 +/‐ 4.7
were the only monosaccharides present in all
the samples as shown in Table 3.2. Ara and
Man were present in a single sample, 5A and 2A, respectively.

3.3.3 Structural analysis

-Samples should be dissolved in deuterated water and then analysed by NMR. Proton NMR spectra

–25–
 can be recorded with a Bruker Avance 400 spectrometer (or similar equipment) operating at a
frequency of 400.13 MHz.
-Samples are first solubilized in deuterated water (D2O) at 343 K. The residual signal of the solvent is
used as the internal standard.
-Proton spectra should be recorded with a 4006 Hz spectral width, 32,768 data points, 4.089 s
acquisition times, 0.1 s relaxation delays, and 16 scans.
-The 1H assignment should be based on 1H-1H homonuclear correlation experiments. That should be
performed with a 4006 Hz spectral width, 2048 data points, 0.255 s acquisition time, 1 s relaxation
delay; 32 to 512 scans should be accumulated.
-Typical spectra of microalgal extracts analyzed by NMR are provided as a reference. The spectra of
standard -glucan isolated from Phaeodactylum (CERMAV) is provided in Figure 3.2. The signal
corresponding to the characteristic protons of the -glucan are annotated. The other spectra
represent samples of -glucan isolated from other production lots of Phaeodactylum.

Figure 3.2 NMR profiles of -glucan isolated from Phaeodactylum, the characteristic proton signal is indicated in the lower
panel (H1 – H6b).

3.3.4 Determination of the protein content of crude microalgae extracts

The Bradford method can be used to determine the protein content of samples and is based on the
properties of Coomassie blue. In its free cationic form, this reagent absorbs light at 465 nm. When it is
mixed in a protein solution, it binds to the proteins present and the aromatic groups cause a
displacement in its peak of absorption to 595 nm (Bradford, 1976). The absorbance at 595 nm is
measured using a spectrophotometer and the method allows an indirect measurement of the protein
content of a solution if a standard curve composed of known concentrations of BSA is measured at
the same time.
-For ddetermination of the protein in cyanobacteria extracts 20 μl of the sample (1 g/l) was added to 1
ml of the Coomassie blue reagent.
-The mixture should then be vortexed and kept for 5 min at room temperature.
-The absorbance is then measured at 595 nm.
-A calibration curve should be prepared at the same time using a series of BSA solutions with
increasing concentrations ranging from 0.02 to 0.20 g/l. The proportional relationship between the
concentration of BSA and the absorption of light at 595 nm permits the determination of the
concentration of protein in the samples.

–26–
3.4 Purification and characterization methods for chrysolaminarin from
microalgae extracts
The general methods for purification of chrysolaminarin are similar irrespective of the microalgae
species.
-In the case of industrial based production, the microalgae can be harvested by continuous-flow
centrifugation or for small scale batch production the whole batch can be collected and centrifuged.
After centrifugation the microalgae biomass should be immediately frozen at -20 ºC and then freeze-
dried and stored until required.
-Crude microalgae extracts can be prepared, and the polysaccharides extracted using the warm-
water method (Caballero et al., 2016), which causes only minor chemical modifications.
-Proteins are precipitated to remove them from the microalgae extract by shifting the pH. Then
centrifuge the samples (30 min at 5,000 rpm, 4°C) to remove cell debris.
-Thereafter, the chrysolaminarin is precipitated by adding absolute ethanol (1 v:v) for 24 h at 4°C.
-The precipitates are collected by centrifugation (30 min at 5,000 rpm, 4°C), frozen at -80ºC and
preserved by freeze-drying.
-The sugar and protein contents are determined for each fraction using the Dubois and Bradford
assays, respectively. Briefly, the chrysolaminarin-enriched crude extract is characterized using the
phenol-sulfuric acid method (Dubois et al., 1956); as an example, extracts containing 47% reducing
sugar and approximately 5% total protein (Bradford, 1976) can be expected.
The molar ratio of the constituent monosaccharides of polysaccharides can be determined with the
Kamerling et al. (1975) method modified by Montreuil et al. (1986). In order to identify and
quantitatively analyze monosaccharides, it is necessary to hydrolyze polysaccharides by
methanolysis to obtain only monomers. Then these glycosidic residues are trimethylsilylated in order
to obtain volatile residues. In this way, they can be identified and quantitatively analyzed by gas
chromatography in methylglycosides O-trimethylsylilated form (as described above in section 3.3).

–27–
Chapter 4. Purification and analysis of microalgae proteins
4.1 Background knowledge
The term proteome and proteomics were introduced by Mark Wilkins and colleagues in early 1990´s
and describes the entire set or complement of proteins that is or can be expressed by a cell, tissue, or
organism-under a particular set of environmental conditions. The proteome is a highly dynamic
system because of complex regulatory systems that control the expression levels of proteins.
Numerous factors contribute to the generation of complex proteomes (i.e. alternative splicing,
assembly of protein complexes with varied compositions, subcellular locations, chemical
modifications, alternative upstream ORFs in mRNA translation, protein interactions). The proteome
adds new information to the genome and transcriptome studies, since most of the functional
information of genes is characterized by the proteome (Harper and Bennett, 2016).
Proteomics refers to the systematic analysis of proteins and is routinely used to: measure absolute
protein levels, survey the dynamic responses of entire proteomes in both space/time, evaluate the
structures of multi-protein complexes, define patterns of reversible post-translational modifications
(Armengaud, 2016). Significant advances have been made in proteomic technology that has now
become truly quantitative and mass spectrometry (MS) continues to be a key tool. Large-scale protein
quantification-identification is being used in a panoply of applications and several proteomes have
been mapped so far. Three main steps are integrated in a general proteomic workflow: sample
preparation, protein separation and protein identification. Sample preparation involves: 1) protein
extraction, in which the method should be adapted to the type of sample (e.g. cells or fluids) and 2)
protein reduction, alkylation and enzymatic digestion (e.g. trypsin digestion) with the aim being to
generate unique peptides suitable for mass spectrometry. For protein separation, the methodology
used could be; gel based (e.g. 1D or 2D -dimensional gel electrophoresis) or gel free (e.g. liquid
chromatography-LC) approaches and both have been developed and utilized in a variety of
combinations to separate proteins. Furthermore, depending on the objective of the proteomic analysis
(qualitative/quantitative), both approaches can be carried out using label or label free protein/peptide
technologies. For protein identification, peptides are analysed using a mass spectrometer (ESI or
MALDI) for detection of each peptide with a unique mass and fragmentation pattern and mass spectra
are matched against protein sequence databases for protein identification. SWATH-MS (Sequential
Window data independent Acquisition of the Total High-resolution-Mass Spectra), is an example of a
modern MS based quantitative label free technology that can combine gel and LC separation of
proteins/peptides.
Microalgae are prokaryote or eukaryote unicellular organisms that can be found in fresh or saltwater.
The high biodiversity and genome plasticity of microalgae makes this “green biomass” a largely
untapped reservoir of novel and valuable bioactive compounds with the potential for industrial
development such as pharmaceuticals, cosmetics, nutritional supplements, molecular probes,
enzymes, fine chemicals, and agrichemicals. They offer clear advantages over terrestrial species, due
to their: fast growing and short generation time, simple manipulation, capacity of adaptation to varied
environmental conditions and huge bio-diversity. Moreover, microalgae are an important reservoir of
novel and bioactive compounds because they are mainly rich in proteins (typically 25–40% of the dry
weight), lipids, carbohydrates followed by pigments, vitamins and minerals. So far, microalgae are
being used and investigated as a source of lipids, secondary metabolite and carbohydrates and
proteins have been largely ignored. Few microalgae proteomic studies exist and the main challenges
in terms of proteomic workflows are related to: 1) the protein extraction, because of the rigid cell wall
and polysaccharides and glycoprotein complexes and 2) data analysis, because of the lack of
microalgae genome and transcriptomes studies available. In the context of the Algae4a-b project, the
aim was to explore the proteome of the microalgae T. chuii in order to develop high-value added
compounds.
The following sections provide a detailed workflow for protein sample preparation for proteomic
analysis of the microalgae T. chuii, but the approach is applicable to other microalgae although due to
the differing characteristic of their cell wall the method for disruption and protein solubilisation will
probably require optimisation. The main workflow is summarized in Figure 4.1.

–28–
 Mechanical Protein quantification/
Hydration Centrifugation
cell disruption extracts quality evaluation

Soluble
protein
extract
Freezed-dried
Biomass
1D SDS-PAGE
(Bead milling) (12%)
Proteomic
analysis

Label free SWATH (Sequential

window-acquisition of all
theoretical mass spectra) –
quantitative

Figure 4.1 Overview of the general experimental workflow used in the preparation of the microalgae T. chuii protein extracts.

4.2 Microalgae Tetraselmis chuii total protein extraction using mechanical

disruption
Sample preparation is one of the most critical steps in proteomic studies and good samples lead to
high quality results. The efficiency of extraction methods that combine cell disruption methods
(mechanical: e.g. bead milling, high pressure and non-mechanical: e.g. enzymatic or osmotic shock)
with protein solubilization solutions always varies with the type of sample. Several cell disruption
methods/solvent solutions are being applied and tested to extract microalgae total protein for
proteomic analysis (Gao 2016; Phong 2018). In terms of biochemical composition, microalgae have
non-protein interference compounds such as lipids, polysaccharides, pigments, and oxidative
enzymes. In addition, they are constituted by a rigid cell wall that confers high mechanical
strength/chemical resistance and they are rich in complex polysaccharides, glycoproteins and
minerals. Therefore, the development of an efficient protein extraction method for microalgae is
required to obtain good quality proteome profiling studies. However, the approach chosen will always
depend of the microalgae strain used (Safi et al., 2014).
After several optimization tests, a protocol was developed for preparation of total protein extracts from
the microalgae T. chuii. Special cares should be taken in protein sample preparation for proteomics in
order to avoid contamination in MS analysis, such as: wear gloves and a lab coat, use protein low
bind eppendorf tubes, use high pure grade reagents, protein preparation should be done in either a
laminar flow hoods or in a clean, low air turbulent environment, and use clean containers/tools.
-Weigh 50 mg of microalgae lyophilized biomass in a 2 ml microcentrifuge tube. Add 1 ml of Milli-Q
water at room temperature. Incubate the microalgae suspension at 4ºC for 30 min in the dark on a
rotary tube mixer for biomass rehydration (Figure 4.2).
-After incubation, add 0.5 ml of the hydrated biomass to a precooled-on-ice 2 ml microtube with cap
containing 300 mg of zirconia/silica beads (0.5 mm). Supplement the suspension with additional 0.4
ml of Milli-Q water at 4ºC. Always keep the hydrated sample on ice.
- Mechanical disruption of hydrated biomass is carried out in a vibrating bead milling. For instance,
Mixer Mill MM400” Retsch®, with a frequency of 30 s-1 and four disruption cycles of 30 s (Figure 4.2).
-Centrifuge the homogenates at 28,000 x g, 4 ºC, for 30 min, and keep the sample on ice (Figure 4.1).
Collect the supernatant with the protein soluble fraction to a new precooled-on-ice microtube of 2 ml.
-To stabilize the proteins and avoid degradation, add a saline solution or Tris buffer pH 8.3, a

–29–
 Protease Inhibitor Cocktail, glycerol. Agitate the extract briefly by vortex and incubate at 4 ºC for 2 h.

Hydration Mechanical Centrifugation Final protein

disruption extract

(Bead milling) (With stabilizing

additives)

Figure 4.2 General appearance of the microalgae T. chuii protein extracts at different steps of the procedure.

Collect an aliquot of 100 µl of each protein sample extracted (Figure 4.2) and store the rest at -80ºC.
To proceed with subsequent concentration determination, a colorimetric assay based on the
Bradford method can be used (Bradford 1976). A number of kits are available and the Quick StartTM
Bradford protein assay kit (Bio-Rad) has revealed to be appropriate for measuring the protein
extracts prepared from microalgae.
The method involves the addition of an acidic dye (Coomassie Brilliant Blue G-250) to a protein
solution; under these conditions, the dye is in the doubly protonated red cationic form (A. max = 470
nm) and, when the dye binds to protein, it is converted to a stable unprotonated blue form (A. max =
595 nm). The dye binds primarily to basic (especially arginine) and aromatic amino acid residues
and it is the blue protein-dye form that is detected at 595 nm using a spectrophotometer. By
comparison to a standard curve prepared from a protein with a known concentration a measurement
of the protein concentration in the sample can be determined.
The protocol described below is for a 96-well microplate assay, using a standard curve of bovine
serum albumin-BSA with a linear range of 0.125–2 mg/ml.
-Remove the 5x dye reagent from the 4°C storage and let it warm to ambient temperature. Mix the 5x
dye reagent a few times before use.
-Plan the design of the 96-well microplate and prepare the BSA standards and samples in triplicate;
the blank should be prepared using the solution in which the protein sample is solubilized.
-Pipette 5 μl of each standard and the blank in triplicate into the multi-well plate.
-Pipette 5 μl of a 1:2 dilution of the microalgae protein extract prepared as described above.
-Dilute the 5x concentrated Coomassie blue dye to 1x concentrated using Milli-Q water at room
temperature and mix well. Determine the total final volume of the Coomassie dye solution required so
sufficient is prepared (multiplying the number of wells used for the assay by 260 μl).
-Add 250 μl of 1x diluted Coomassie blue dye solution to wells containing sample/standard/blank
using a multichannel pipette. Mix slightly but avoid introducing bubbles that will interfere with
spectrophotometric reading.
-Incubate the plate in the dark for 5 min at room temperature and then read in a microplate reader set
at the appropriate wavelength (595 nm for protein).
-Perform data analysis:
1) Calculate the average reading obtained for the blank and subtract the value from all the readings
obtained for the standards and unknown samples.

–30–
 2) Create a standard curve by plotting the 595 nm values (y-axis) versus their concentration in
mg/ml (x-axis).
3) Determine the unknown sample concentration using the absorbance value and reading off the
corresponding concentration from the standard curve. If the samples were diluted before protein
determination, adjust the final concentration of the unknown samples by multiplying by the dilution
factor used.

4.3 Microalgae T. chuii proteome profile analysis by 1D PAGE

The quality of the total protein extractable from microalgae can be assessed by one-dimensional (1D)
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This technique was first
described by Laemmli (1970) and describes a discontinuous and denaturing polyacrylamide gel
system in which the proteins migrate in response to an electrical field through pores in a
polyacrylamide gel matrix, with pore size decreasing with increasing acrylamide concentration. SDS-
PAGE is routinely used to separate proteins and the combination of pore size and protein charge,
molecular weight, and shape determines the migration rate of the protein. Check of the purity of
protein samples, estimation of molecular weights of unknown proteins, separation of complex
mixtures of proteins, and monitoring of protein expression profiles are few examples of the
applicability of this technique.
The protocol described below is to assess the proteome profile of microalgae T.chuii using a vertical
gel electrophoresis system (for instance, Mini-PROTEAN Tetra Cell, Bio-Rad). Standard mini-gel (8 x
10 cm) SDS-PAGE of 12% polyacrylamide with 0.75 mm of thickness and a comb of 10 wells are
used. The protocol is directed for two mini-gel preparation. Respect the safety rules and consult the
safety data sheets (MSDS) of each reagent prior to use, e.g., acrylamide (CAS nº 79-06-1) as a
monomer is considered toxic, directly affecting the nervous system, and it may reasonably be
considered to be a carcinogen. Acrylamide is readily absorbed through intact skin from aqueous
solutions. Wear gloves and laboratory coat when preparing the polyacrylamide gel solution.

4.3.1 1D SDS-PAGE gel preparation

-Remove the reagents (40% acrylamide/Bis Solution 37.5:1 (Bio-Rad), 1.5 M Tris-HCl pH 8.8, 1 M
Tris-HCl pH 6.8) from the 4°C storage and let it warm to room temperature.
-Clean and completely dry glass plates, combs, and spacers are required.
-Prepare the gel assembly chamber in the respective system according the instructions of the
manufacturer. Test the system with water in advance to prevent loss of solution.
-Prepare the resolving and stacking gel solutions following the order of reagents indicated in Table
4.1.
-Advance first with the preparation of the resolving gel by mixing the reagents in a glass Erlenmeyer
with a magnetic stirrer and agitate the solution slowly while adding the reagents to avoid bubble
formation.
-Degas the mixture of the first three reagents added (H2O, acrylamide, 1.5 M Tris-HCl, pH 8.8) for 10
min with agitation using a vacuum pump, this step is recommended but not essential but it will
increase the gel resolution by removal of dissolved oxygen.
-Proceed with the addition of SDS (sodium dodecyl sulfate), APS (ammonium persulfate) and TEMED
(N,N,N′,N′-Tetramethylethylenediamine). The APS should be made fresh immediately before use
and the TEMED should be added immediately before pouring the gel; both reagents are initiators of
the polyacrylamide polymerization.
-After mixing, transfer 3.5 ml of gel solution by using a 5 ml pipette to each casting chamber between
the glass plates and fill up to about 0.7 cm below the bottom of comb when the comb is in place.
Add a small layer of ddH2O to the top of the gel prior to polymerization to straighten the level of the

–31–
 gel and wait for gel polymerization (around 20 min).
-Once the gel has polymerized, start to prepare the stacking gel (5%) by adding the solutions
indicated in Table 4.1.
-Follow the same procedure for the preparation of the stacking gel as described above for the
resolving gel.
-Remove the layer of ddH2O using filter paper and transfer 1.5 ml of the stacking gel solution by using
a 1 ml pipette to each casting chamber between the glass plates, fill up until it reaches the top.
-Insert a 0.75 mm ten wells comb in the glass-cassette, wait for gel polymerization and then carefully
remove the comb.

Table 4.1 Volume of reagents to be used for the 12% SDS-PAGE gel preparation.

Resolving gel Stacking gel

Solution reagents (12%) (5%)
V (ml) V (ml)
ddH2O 4.296 3.640
40 % Acrylamide mix 3 0.625
1.5 M Tris-HCl, pH 8.8 2.5 ---
1 M Tris-HCl, pH 6.8 --- 0.630
10 % (m/v) SDS 0.1 0.05
10 % (m/v) APS 0.1 0.05
TEMED 0.004 0.005
Final volume (ml) 10 5

4.3.2 1D SDS-PAGE microalgae protein sample preparation

-Mix 6 μg of total protein from microalgae T. chuii extracts prepared above with 2x SDS loading buffer
(100 mM Tris-HCl pH 6.8, 4% SDS, 20 % Glycerol, 0.2% Bromophenol Blue, 200 mM DTT) in order
to have 1x SDS loading buffer in the final sample. It is advisable to dilute the 6 μg of total protein in
50 mM of Tris buffer pH 8.3/20% glycerol to fix the same volume for all samples (e.g. 10 μl) and
then add 2x SDS loading buffer. Mix the components with a pipette.
-Proceed for protein heat denaturing by boiling the samples at 95ºC for 5 min in a dry bath.
-Centrifuge the samples at 14,000 x g at room temperature for 3 min and leave the samples at room
temperature until you are ready to load them onto the gel.

4.3.3 Electrophoresis
-Use the prepared SDS-PAGE gels and introduce the glass-cassette inside the electrophoresis
chamber (following the instructions of the manufacturer).
-Fill the upper and lower buffer chamber with 1x SDS-PAGE running buffer (25 mM Tris-HCl, pH 8.3;
192 mM Glycine; 0.1 % SDS). Fill in first place the inner chamber and wash the sample wells with
running buffer.
-Load the total volume of denatured and reduced protein samples on the wells of the gel (one
sample/well) and also 4 μl of molecular weight marker (for instance, PageRuler™ Plus Prestained
Protein Ladder, Thermo Scientific).
-Connect the electrophoresis tank to the power supply (for instance, Power Pack Universal Power

–32–
 Supply, Bio-Rad) and run the gels at constant 30 mA/gel until the migration dye reaches the bottom
of the glass-cassette (take around 90 min). The electrophoresis should run at 4ºC in a refrigerated
chamber to avoid overheating and bad gel resolution.
-After electrophoresis, remove the gel glass-cassette from the electrophoresis apparatus and remove
the gel from the glass-cassette. Wash the gel with water for 5 min and then proceed for gel staining.

4.3.4 Protein detection

The protein staining method should be selected taking into consideration the protein concentration
loaded onto the SDS-PAGE gel. The staining procedures have different sensitivities, typically
Coomassie blue (CBB) staining allows detection in a minimum of 30-100 ng (depending on the stain
characteristics eg. Colloidal or not) and the more sensitive silver staining method can detect as little
as 5-10 ng. To visualize the proteome profile of microalgae T. chuii the two methods of staining can
be carried out. The CBB method is firstly applied in order to assess the separated proteins. If this is
adequate then the silver staining method can be applied for better detection of the full proteome
profile.
-Incubate the gels on 50 ml/gel of CBB staining solution (40 % Methanol, 10% Acetic acid, 0.1 %
Coomassie Brilliant Blue R-250 in ddH2O) for at least 1 h at room temperature with slightly constant
shaking.
-Destain the gels by incubation on distaining solution (40 % Methanol, 10 % Acetic acid, ddH2O) at
room temperature with slightly constant shaking.
-Change the distaining solution once saturated as often as necessary until protein bands are visible.
-Take a picture of the gel in a gel imager documentation system and analyse the results.
-Completely destain the Coomassie stained gels by incubation with constant shaking at room
temperature in 50% Methanol/50 mM Ammonium bicarbonate solution. This process must be done
until the bands disappear completely.
-Wash the gels 3x during 20 minutes in Milli-Q water with constant shaking at room temperature.
-The silver staining procedure is carried out using the Silver Stain PlusTM Kit (Bio-Rad) following the
manufacturer’s instructions.
-Take a picture of the gel in a gel imager documentation system and analyse the results.

4.4 Microalgae T. chuii sample preparation for SWATH quantitative proteome

analysis
Quantitative proteomic analysis of soluble protein extracts from microalgae T. chuii is carried out
using SWATH-MS (Sequential Window data independent Acquisition of the Total High-resolution-
Mass Spectra) technology. SWATH‐MS method is a label‐free technology and is a particularly
promising quantitative method for routine high‐throughput screening due to its ability to combine the
protein identification data obtained from shotgun proteomics with data‐independent acquisition to
accurately quantify large numbers of peptides (Gillet et al., 2012). The technological procedure
followed for LC-SWATH-MS acquisition on a Triple TOF™ 5600 System (Sciex®, Framingham, MA)
using two modes of data acquisition, information-dependent acquisition (IDA) for protein identification,
followed by SWATH acquisition for protein quantification is described in Anjo et al. (2015) and Santa
et al. (2016). The protocol for protein sample preparation to be analysed by SWATH-MS is described
below.
-Defrost the protein extracts on ice and separate 200 μg of total protein in a 1.5 ml microtube.
-Supplement the separated protein solution with 100 mM DTT and 1.7% SDS and mix well with the
pipette.
-Boil the homogenates at 95 ºC for 10 min and then allow to cool at room temperature for complete

–33–
 thermal denaturing.
-Add 1 μl of 40 % acrylamide solution per each 15 μl of protein solution for protein alkylation.
-Keep the protein samples at -80ºC until SWATH analysis. Protein samples are de novo quantified
and about 50 μg of each sample is used.

–34–
Chapter 5. Molecular methods
5.1 Extraction of total RNA from human and fish cells
Gene expression is the process where the information contained in the genes inside the nucleus is
copied to a RNA molecule and used to synthesize a functional gene product in the cytoplasm.
Therefore, characterization of the RNAs from cells or tissues reflects the genes that are activated and
enables us to infer about their relative abundance and importance in physiology. Gene expression
studies on both normal and diseased skin have contributed to elucidate the molecular repertoire of the
different skin cell types and cellular interactions. In the cosmetic industry characterization of the genes
expressed by the skin and that regulate stress, aging, cell proliferation and growth are of interest.
Below we present a step-by-step protocol to study gene expression in human dermal fibroblasts to
gain further insights on the molecular pathways that govern human skin physiology. Fibroblasts are
the main cells of dermis which is made of two layers of connective tissue an interconnected mesh of
elastin and collagenous fibers.
Briefly, tissues or cells (frozen or preserved in RNA-later solution) are exposed to mechanical
disruption in lysis buffer (a chaotropic agent that contains high concentration of guanidine
isothiocyanate solution with 1% -mercaptoethanol) to denature proteins and protect nucleic acids
from degradation. The RNA molecule in solution is isolated from genomic DNA, proteins and
tissue/cell debris using a series of centrifugations and ethanol precipitation/washing steps. DNase is
added to obtain RNA free of genomic contamination.
RNA handling requires a RNase-free environment and special care to avoid RNA degradation is
essential. All material should be autoclaved (121ºC, 20 min,1 atm relative pressure) or alternatively
materials and working surfaces should be cleaned with commercial chemical agents to inactivate
RNases. Clean and disposable gloves should be worn at all times. RNA samples should always
remain on ice during downstream applications. The procedure described below is based on the
protocol for the animal tissue RNA purification kit from Norgen (Canada) that uses resin columns to
sequestrate and purify RNA from a solution.
-Add 300 µl of lysis buffer to the cells. Transfer the lysate with the aid of a pipette to an RNase-free
microcentrifuge tube and add 600 µl of RNase-free water and vortex.
-To pellet cell debris, centrifuge the lysate for 1 min and then transfer the supernatant to a new
RNase-free tube. Add 450 µl of 96-100 % ethanol to the lysate and vortex to mix.
-Specific columns are used to bind and isolate RNA. Assemble the resin columns in the specific
collection tubes and add 650 µl of the cell lysate and centrifuge at 14,000 x g for 1 min. The flow
through is discarded.
-Reassemble the spin resin column in the collection tube and add 400 µl of the wash solution and
centrifuge for 2 min.
-Discard the flow through and assemble the resin column in a new collection tube.
-Add 100 µl of DNase Incubation Buffer and 15 µl of DNase I enzyme (2 U/µl) to the resin column and
centrifuge at 14,000 x g for 1 min. Add back the flow through in the column and incubate at room
temperature for 15 min.
-Add 400 µl of wash solution (chaotropic salts in ethanol solution) to the column and centrifuge for 1
min to remove impurities such as protein and polysaccharides from the resin column.
-Discard the flow through and wash twice. Spin the column for 2 min to dry the resin.
-Place the column in a clean microcentrifuge tube and add 50 µl of RNase free water or TE buffer for
the elution of RNA to the column.
-Centrifuge for 2 min at 200 x g, followed by 1 min at 14,000 x g.
The purified RNA sample can be stored at -20°C for a few days but at -70°C for long term storage.

–35–
5.2 Synthesis of cDNA
Complementary DNA (cDNA) is an artificial DNA molecule synthesized in vitro using as template the
single stranded RNA. This reaction is catalyzed by a reverse transcriptase enzyme that promotes the
conversion of RNA to DNA by a process termed reverse transcription. The cDNA molecule can be
used directly as a template for the Polymerase Chain Reaction (PCR). In many applications, oligo(dT)
or random primers can be used to prime cDNA synthesis. Oligo(dT) primers are used to amplify
mRNA and random primers are used for synthesizing cDNA from input RNA because all classes of
RNA are amplified (eg. rRNA, tRNA) including the RNA target molecules. A combination of oligo(dT)
and random primers provides a better representation of full-length product. In alternative, if the target
gene is lower expressed cDNA can be produced using gene specific primers. In this section,
synthesis of cDNA from total RNA isolated from human fibroblasts using the iScript cDNA synthesis
Kit (Bio-Rad) is outlined. The reactions are performed on PCR microtubes (0.2 ml) and incubations at
different temperatures in a thermocycler (Bio-Rad). To verify the quality and integrity of the cDNA
produced, amplification of a housekeeping gene is used (Table 5.1). This step is performed prior to
quantify target gene expression.
-To 100 fg-1 µg of total RNA add 4 µl of the 5x iScript Reaction mix, 1 µl of iScript reverse
transcriptase, and complete the volume with nuclease free water up to 20 µl.
-Incubate 5 min at 25ºC to prime and 20 minutes at 46 °C for the reverse transcription reaction to
occur. Subsequently incubate 1 min at 95ºC to inactivate the enzyme.
-The cDNA produced can be stored at 20ºC until further use.

5.3 Quantitative gene expression: PCR platform design and analysis

Quantitative Polymerase Chain Reaction (RT-qPCR) is the method of choice to characterize transcript
profiling. This is a highly sensitive, accurate and reproducible technique, ideal for small to medium
scale targeted transcriptomic analysis. Target cDNAs are amplified using gene specific primers that
are designed based on the nucleotide sequence of the target gene. The amplified products are in
general between 70-200 bp long.
5.3.1 Primer design
Primer length should be 18-22 bp, with a melting temperature between 60-63ºC and a GC content of
around 40-60% to ensure maximum product stability. Several programs to aid on the design of
primers pairs are available (e.g.: primer express, Primer Premier, NCBI primer-BLAST).
5.3.2 Reaction preparation
The qPCR reaction performed used the SYBR Green dye chemistry, which binds to the forming
double-stranded DNA template, and the DNA-dye complex absorbs blue light (λmax = 497 nm) and
emits green light (λmax = 520 nm). The reaction reagents used are from Bio-Rad and amplification
reactions were performed in a thermocycler cfx-connect (Bio-Rad). For a 10 μl reaction the qPCR
reaction was as follows: 1 μl of cDNA, 4 μl of primers (0.5 μM), 5 μl of 2x SYBR Green dye. Usually a
RT-qPCR typical cycle consists of a 95 °C for 10 min initial denaturation step, followed by a cycling
reaction of 40 cycles of 95 °C for 15 s and an annealing temperature of 60-62 °C for 1 min. The
primer specificity of the PCR reaction and the potential formation of primer-dimers are monitored by
dissociation curve analysis and double checked on 2% (w/v) agarose gel electrophoresis. To
characterize changes in relative gene expression of target genes, normalization of the data using at
least two reference genes is carried out. Reference genes have a constant relative expression level
under normal and experimental conditions. Housekeeping genes are generally used as referenced
genes to normalize gene expression. A list of reference genes used to normalize expression data in
human cells have been identified, with beta actin beta (ACTB) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) proven to be the most stable (Table 5.1).
5.3.3 Data analysis

–36–
For the relative quantification of target gene expression, a modification of the comparative threshold
cycle (Ct) method is used. The ratio of the target gene transcripts (X) in the sample of interest (S),
normalized to ACTB, and the target gene transcripts in different treatments (R), is calculated as (1+E)-
∆∆Ct
, where ∆∆Ct is calculated as [(CtX.S-CtACTB.S)-(CtX.R-CtACTB.R)]. PCR efficiency (E) for each
amplicon is calculated employing the linear regression method on the Log(Fluorescence) per cycle
number data, using the LinRegPCR software. Then, same calculations are performed for GAPDH as
a second reference gene. Finally, the geometric average of the relative gene expression against
ACTB and GAPDH is determined in every sample of the different treatments.

Table 5.1. Reference gene candidates for qPCR analysis of human dermal cells.

Name Gene Accession No. Function Reference

symbol

Actin beta ACTB NM_001101.3 cytoskeletal protein Li et al., 2011

Glyceraldehyde-3- Li et al., 2011
phosphate dehydrogenase
GAPDH NM_002046.3 glycolytic enzyme
Glucuronidase beta GUSB NM_000181.3 Hydrolase that degrades Li et al., 2011
glycosaminoglycans
TATA box binding protein TBP NM_001172085.1 transcription factor IID Li et al., 2011
(TFIID)
Succinate dehydrogenase SDHA NM_001294332.1 subunit of succinate- Li et al., 2011
complex ubiquinone
oxidoreductase
Hypoxanthine HPRT1 NM_000194.2 transferase enzyme Li et al., 2011
phosphoribosyl
transferase-1
Tubulin, alpha 1a TUBA1A NM_006009.2 component of Li et al., 2011
microtubules

Tubulin, beta 1 TUBB1 NM_030773.2 component of Li et al., 2011

microtubules

Vimentin VIM NM_003380.2 structural protein Li et al., 2011

5.4 Gene expression analysis in fish larvae and tissues

The following section outlines how to prepare RNA from fish for gene expression analysis and the
quantitative PCR method that can be used (this section is linked to section 7).
5.4.1 Total RNA isolation from embryos and larvae
-Sample preparation and homogenization
As a general rule, all samples should be managed using gloves and sterile material double
autoclaved. Tissue homogenization can be done using several procedures. In this protocol, the lysing
matrix D (Q-Bio-Gene) is found to be a very fast and highly efficient method for disruption of the
tissues.
 Tubes are labelled and 1 ml of Trizol or Trisure reagent added.
 Then, samples of larvae or embryos are taken out from RNAlater solution and the
appropriate number of larvae/embryos (~25-30 embryos or just recently hatched larvae) is
placed into the lysing matrix tube using a sterilized spatula.
 To homogenize the samples, the tubes are located in the Fast-prep FG120 instrument
(Bio101) and disrupted at speed setting 6 and incubated at room temperature (RT) for 5
min.
-Phase separation

–37–
  A volume of 200 µl of chloroform is added to each tube and mixed by inversion.
 After incubating at RT for 2 min, the samples are centrifuged at 12,000 x g for 15 min.
 Then, the upper aqueous phase is transferred into a new 1.5 ml tube taking care not to
disturb the interphase.
-DNase treatments (based on ISOLATE II RNA Mini kit from Bioline)
 Adjust RNA binding conditions. The homogenized sample is added to the same volume of
Lysis Buffer and absolute ethanol and mixed well by pipetting (e.g., if the phase volume
recovery is 500 µl, add 500 µl of lysis buffer and 500 µl of absolute ethanol).
 Load the volume into the column and centrifuge for 1 min at 11,000 x g. (750 µl max
volume). Repeat this step to load all the volume.
 Wash the membrane (optional step) by adding 350 μl Membrane Desalting Buffer and
centrifuge at 11,000 x g for 1 min to dry the membrane.
 Remove DNA by adding DNase I reaction mixture (10 μl reconstituted DNase I to 90μl
Reaction Buffer for DNase I). Add 95 μl DNase I reaction mixture directly onto the center of
the silica membrane and incubate at RT for 30 min.
-Wash and dry silica membrane
 Add 200 μl Wash Buffer RW1. Centrifuge for 1 min at 11,000 x g. and change the collection
tube.
 Add 600 μl Wash Buffer RW2 (ethanol 70%). Centrifuge for 1 min at 11,000 x g. Discard
flow-through and place the column back into the collection tube.
 Add 250 μl Wash Buffer RW2 (ethanol 70%). Centrifuge for 1 min at 11,000 x g. Discard
flow-through and place the column back into the collection tube.
 Centrifuge again at 11,000 x g for 2 min to dry the membrane.
-Elute RNA. Place the column into a nuclease-free 1.5 ml tube and add 60 μl RNase-free water to the
column, incubate for 1 min at RT and centrifuge at 11,000 x g for 1 min.
-Quantify RNA samples using a NanoDrop and determine the integrity by electrophoresis on an
agarose gel at 2%. The ratio 260/280 should be 2.1-2.2 to be considered of high quality. Also, load
1-5 µl on an agarose gel to check the integrity of 18S and 28S bands (Figure 5.1).
- Clean-up DNAase treatment. If quantity and quality of RNA are optimal, repeat sample treatment
with DNase I. Bring the volume up to 100 μl with RNase-free water. Add 300 μl Lysis Buffer RLY
and 300μl of absolute ethanol and repeat the procedure. Then, samples are stored at -80ºC until
use.

–38–
Figure 5.1 Top panel, Agarose gel electrophoresis of extracted larvae RNA. Two main bands (28S and 18S are visible).
Lower panel, NanoDrop results giving an example of the quality and yield of RNA.

5.4.2 RT-qPCR
-cDNA synthesis
Before starting, two thermal blocks at 42 ºC and 85ºC should be prepared. Total RNA is
retrotranscribed into cDNA using iScript cDNA synthesis kit (Bio-Rad). This kit uses a blend of
oligo(dT) and random hexamer primers.
 Put into a 1.5 ml tube the volume of RNA sample corresponding to 1 µg
 Add 4 µl of 5x iScript reaction mix and 1 µl of iScript reverse transcriptase for each reaction
(prepare a mix for all the reactions to do)
 Add RNase-free water to reach a final volume of 15 µl
 Incubations:
- 5 min at 25ºC
- 30 min at 46ºC
- 1 min at 95ºC
 Hold at 4ºC
 Make 1/10 dilution of cDNA samples in DEPC water and store at -20ºC until use
-qPCR assays
All qPCR assays are carried out in the same way irrespective of the experiment. Melting temperatures
for all primers are similar.
 Add 4 µl of cDNA sample into a 500 µl tube. This cDNA quantity corresponds to 10 ng of
original RNA template for each reaction. Reactions are always carried out in duplicates (2 µl
of cDNA per reaction)
 Prepare a reaction mix for all the samples (10-µl final volume of each reaction) according
with the following ratios:
 0.4 µl of a mix of forward and reverse primers (10 µM each)
 5 µl of SYBR Premix Ex Taq (Takara)
 2.6 µl of water
 Add 16 µl of the previous mix to the tube with cDNA (8 µl per reaction)
 Distribute in a 96-well plate (Bio-Rad), 10 µl per well (in duplicates)
 Centrifuge the plate 3 min at 3,000 x g
 Cover the 96-well plate with an adhesive seal and put in the CFX96TM Real-Time System
(Bio-Rad).
 The amplification protocol used is as follows: initial 7 min denaturation and enzyme
activation at 95ºC, 40 cycles of 30 s at 95ºC, 15 s at 68 °C (annealing temperature will
depend on the melting temperature of the primer pair designed for the target gene) and 30 s

–39–
 at 72 °C. Finally, a melting curve is recorded heating to 95°C and then cooling slowly at
0.5°C/s until 70°C, to check the specificity of the amplification.
 To analyze the data, the Ct values are normalized to the geometric mean of reference
genes (ubi and actb2). Relative mRNA expression is determined using the comparative
method 2-(∆∆Ct).

5.5 Sampling of biological samples for microbiome analyses

It is now recognized that the community of microbes that inhabits the bodies of multicellular
organisms (their microbiomes) can greatly influence their physiology in health and in disease, as
revealed by several large projects such as the Human Microbiome Project or the Ocean Sampling
Day campaigns (Goodrich et al., 2014). Global microbiome characterization studies are still scarce in
fish but the skin and the intestine, which are important innate immune barriers influenced by changes
in the environment or food, appear to harbour a substantial diversity of microbes that begin to take
shape from early ages (Egerton et al., 2018). This chapter presents the protocols used in the
Algae4a-b to identify the bacterial communities on the surface of fish larvae and juveniles or adult fish
skin or gut tissues (Pinto et al., 2019), using 16S rRNA gene metagenomics, and to evaluate the
effects of exposure of fish to microalgae.
Samples need to be collected and preserved in aseptic conditions, so that the obtained microbiomes
accurately reflect the original conditions of the sampling at the timing of collection and are not
contaminated or altered by microbial growth or nucleic acid degradation during sampling, transport or
storage.
The sampling procedure is usually carried out using disposable gloves, sterile sampling containers
and solutions and sterilized or disposable dissection material. When possible, sample collection is
carried out in a laminar flow cabinet or by a flame or, when not possible, the sample is rapidly
collected into the sterile sampling container and sealed. Common preservation methods include
sampling into commercial bacterial preservation solutions or in RNAlater with subsequent freezing at
the temperatures recommended by the supplier or direct freezing using dry ice or liquid nitrogen
followed by storage at −80°C. It is important to be consistent in the collection and storage between
samples and to keep conditions constant, for example, avoiding inconsistent freeze-thaw cycles. The
procedure described below is used for the collection of samples from Senegalese sole (S.
senegalensis) larvae or juvenile/adult individuals, for further bacterial microbiome analyses.
-For the sampling of S. senegalensis, anesthetise and transfer the animals (larvae or juvenile sole) to
a laminar flow cabinet and carefully rinse them in sterile water.
-Directly transfer multiple larvae to 10 ml of RNAlater in sterile polypropylene tubes of 15 ml, taking
care not to exceed the 1:5 vol/vol ratio of sample to RNAlater. Incubate samples at 4ºC overnight
and then store at -20ºC until DNA extraction.
-In alternative, dissect juvenile or adult sole to excise a sample from dorsal skin and from the anterior
gut. Incubate in RNAlater at 4ºC overnight and store at -20ºC until DNA extraction.

5.5. Extraction of total DNA for bacterial microbiome characterization

The procedure uses the DNeasy Blood & Tissue Kit (Qiagen) for the extraction of total DNA from the
fish samples, with some modifications to increase the extraction of bacterial DNA, in order to prepare
the starting material for bacterial microbiome characterization. This commercial kit is based on the
enzymatic and mechanical lysis of the tissues or cells in a lysis buffer followed by digestion with
proteinase K. The lysate is then purified using DNeasy Mini spin columns (with silica-based
membranes), followed by multiple washing steps and elution of the DNA sequestered in the columns.
Modifications to the original protocol include the pre-digestion with lysozyme to lyse Gram-positive
bacteria, mechanical disruption with 0.1 mm zirconia/silica beads to disrupt the bacteria cells and the
optional RNase treatment to avoid contamination of the total DNA with RNA.

–40–
5.5.1. Lysis of fish samples
-Label one tube per sample, using 2 ml screw-cap microcentrifuge tubes.
-Prepare and pre-weight one lysis tube per sample, after adding to the tubes 200 µl of lysis buffer (20
mM Tris-HCl pH 8.0, 2 mM sodium EDTA, 1.2% Triton X-100, 40 mg/ml lysozyme), 200 µl of AL
buffer (supplied with the DNeasy kit), and ~400 mg of glass beads (0.1 mm zirconia/silica beads,
Biospec) or two iron beads (5 mm iron beads, Qiagen). These steps are performed in a laminar flow
cabinet.
-Add 3 fish larvae to the tube and register sample weight. Perform the mechanical disruption (for
instance, in a Qiagen Tissue Lyser II for 3 x 5 min at 25 Hz) with the glass beads.
Or:
-Add to a lysis tube one fragment of skin or gut from juvenile sole, excised in a laminar flow cabinet
using sterilized dissection material, and register the weight. Respect the limit of maximum 30 mg of
tissue recommended by the Qiagen kit.
-Perform the mechanical disruption in a Qiagen Tissue Lyser II for 3 x 30 s at 25 Hz with the two iron
beads. Remove the beads, add the glass beads and disrupt for 3 additional periods of 5 min at 25
Hz.
-Incubate disrupted samples for 30 min at 37ºC.
-Add 25 µl of proteinase K (20 mg/ml) and incubate for 30 min at 56ºC.
-Centrifuge tubes at 8,000 rpm to pellet the beads and pipette the lysate solutions to new tubes.
-Add 10 µl of RNase (10 mg/ml), incubate for 10 min at room temperature and add 0.5 volumes of
100% ethanol.
-Proceed to the column purification protocol (see below).
5.5.2 Column purification of the DNA
-Transfer the extraction solution with ethanol to one Mini spin column placed in a 2 ml collection tube,
both provided in the DNeasy Blood & Tissue Kit. Spin for 1 min at 6,000 x g in a bench centrifuge and
discard the flow-through (FT).
-Add 500 µl of wash buffer AW1, centrifuge 1 min at 6,000 x g and discard the FT.
-Add 500 µl of wash buffer AW2, centrifuge 4 min at 20,000 x g and discard the FT. Repeat the
centrifugation for an additional 1 min to dry the membrane.
-Place the Mini spin column in a clean 1.5 ml microcentrifuge tube and pipet 50 µl of elution buffer EB
(10 mM Tris pH 8.0) directly onto the membrane.
-Incubate at room temperature for 1 min and centrifuge for 1 min at 6,000 x g to elute the DNA
sample.
-Quantify 1 µl of the DNA in a Nanodrop spectrophotometer to verify concentration and purity and run
~100-200 ng by gel electrophoresis in an 1% agarose gel to analyse integrity.

5.6 Construction and sequencing of microbiome libraries

-Construction of 16S microbiome libraries following the protocol “16S Metagenomic Sequencing
Library Preparation - preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq
System” from Illumina, starting with ~100 ng DNA.
-Amplicon-based microbiome studies were used for the identification and taxonomic classification of
specific group of microorganism. Microbiota composition were profiled by sequencing the
hypervariable V3-V4 regions of the 16S rRNA gene. it was PCR-amplified with universal primers S-
D-Bact-0341-b-S-17 primer (forward 5’-CCTACGGGNGGCWGCAG-3’) and S-D-Bact-0785-a-A-21
primer (reverse 5’-GACTACHVGGGTATCTAATCC-3’) (klindworth et al. 2013) designed for dual

–41–
 indexing following the Illumina 16S Metagenomic Sequencing Library Preparation protocol (Part #
15044223 Rev. B).
- Amplification was performed in two steps: i) In a first step, primary PCR captured the V3-V4 region
and it is added a universal adaptor in order to allow the secondary PCR step. ii) in a second step,
secondary PCR was performed using primers with the universal tail, and illumina dual index
sequences and the sequencing adaptors.
-PCR cleanings were carried out using Ampure XP and measured using Quant-iT PicoGreen
(Invitrogen) and pooled equimolarly.
-Sequence 16S reads using an Illumina MiSeq instrument, to obtain a total of 20-30,000 sequences
per sample in a combination of 250-300 bp paired end reads.
-Bioinformatic analysis for sequence data cleaning, removal of chimeras, diversity analyses and
identification and classification of OTUs using in-house or open source software for metagenomics,
such as Qiime (http://qiime.org/) or Qiime2 (https://qiime2.org/), choosing the NCBI 16S rRNA
database for taxonomic assignments with a minimum cut-off at 97% identity (Codoñer et al., 2018).

5.7 Microorganism-specific quantitative PCR

Since the microbiome profiles obtained from the 16S libraries are often qualitative, giving the
proportion of a given microorganism genus or species in percentage relative to the total microbiome, it
is often useful to validate the microbiome results by performing microorganism-specific qPCR. The
following procedure briefly describes how to quantify the load of bacteria from the genus Vibrio, a
common component of the fish microbiota in aquaculture, using genus-specific primers (Tall et al.,
2012).
The optimized qPCR reaction uses the EvaGreen dye chemistry, intercalating in double-stranded
DNA template with increased fluorescence compared to SYBR Green.
-Reaction reagents are from Bio-Rad, using 10 μl reactions containing 300 nM of each primer, 5 μl of
2x SsoFast EvaGreen Supermix (Bio-Rad) and 10 ng of total DNA from each fish sample.
-Cycling conditions, carried out in a StepOnePlus qPCR thermocycler (Applied Biosystems) are: 30 s
at 95ºC followed by 40 cycles of 5 s at 95ºC and 10 s at the optimized annealing temperature (58ºC)
for the Vibrio genus-specific primers, followed by a final melt curve between 65 and 98ºC.
-Specificity of the qPCR reactions should be confirmed by the presence of single peaks in the melt
curves, amplification of expected size bands in 2% agarose gel electrophoresis and sequencing of
the qPCR products.
-In each quantification plate, one standard curve relating amplification cycle with initial template
quantity is generated using serial dilutions of the specific amplicon for the Vibrio genus, cloned in
pGEM-T Easy. Copy number of the Vibrio amplicon is calculated using the following equation:
number of copies = (X ⁄ NA)/(Y x 1×109 x 650), where X is the initial template amount (ng of the
amplicon fragment), NA is Avogadro’s number, Y is the template length (bp of the amplicon), and
650 (Da) is the average weight of a bp (Martyniuk et al., 2009).

–42–
Chapter 6. Protocols for nutraceuticals administration
6.1 Background knowledge
Microalgal extracts are able to trigger a plethora of physiological effects in fish. However, their
functional properties are highly dependent on the method of preparation, dose, route and effective
delivery in target tissues. In aquaculture, three main approaches are used to deliver nutraceuticals
and the method of choice depends on the main objective: a) injection in order to enhance immune
response during vaccination actions; b) immersion specially in larvae to reprogram the methylome; c)
by oral administration through the food providing the active compounds as crude extracts or
encapsulated to increase the half-life and target actions in the intestine. As indicated above, the new
methods to design effective vaccines are progressively incorporating new adjuvants with
proinflammatory properties with high potential to enhance cytokine production. They are considered
as extremely useful to stimulate and reinforce chemotaxis and antibody production against pathogen
antigens (Tafalla et al., 2013). The immersion approach is the most common strategy in larvae and
can be used to exploit their high developmental plasticity since the handling is straightforward and
feasible in an aquaculture setting (Carballo et al., 2018b; Firmino et al., 2017). Finally, the oral
delivery of nutraceuticals is the most used pathway of nutraceuticals in aquaculture for all fish stages.
However, appropriate formulation and encapsulation of components are required to be cost-effective,
preserve the functionality and control the targeted tissues in the organisms. The optimization of these
administration methods and the establishment of a correct strategy according to the nature of the
extract and fish physiology are essential for successful enhancement of production traits in
aquaculture.
For the development of protocols for aquaculture, the flatfish Senegalese sole was selected as the
model (Benzekri et al., 2014; Manchado et al., 2016). This species is nowadays intensively cultivated
in Southern Europe and there exist substantial investments in production of the species, which
demands optimized husbandry and nursery protocols so it can develop into a highly competitive
industry. Previous studies described some effects when sole larvae were exposed to physical stimuli
(Carballo et al., 2018b; Firmino et al., 2017). However, additional routes need to be thoroughly
explored towards the development of new commercial products based on microalgae for the
aquaculture sector.

6.2 Administration of microalgal extracts by injection

A protocol for the administration by intraperitoneal injection of microalgal extracts from
Phaeodactylum tricornutum enriched in polysaccharides to sole juveniles was developed. The results
from the studies are reported in Carballo et al. (2018).
6.2.1 Preparation of microalgal extract and cytotoxicity test
The P. tricornutum strain is initially grown indoors using autoclaved seawater (salinity 33 psu)
enriched with filter-sterilized f/2 nutrients in 50 mL flasks bubbled with filter-sterilized CO2-enriched air
(2%). Microalgae cultures are out in a temperature-controlled room (22ºC) under continuous
illumination using artificial daylight fluorescent lights (150 μmol
Figure 6.1 m−2 s−1). Cells are harvested by continuous-flow centrifugation
Chrysolamin
arin-
4 h after sunrise, and then they are frozen at -20 ºC and
enriched freeze-dried until required for experiments.
microalgae
extract (left) The microalgal-enriched crude extract is prepared using the
and warm-water extraction method (described in chapter 3, Chiovitti
Yestimun
(right) used et al., 2004) with slight modifications. After collecting the
for the precipitates by centrifugation, they are frozen at -80ºC and
testing. preserved by freeze-drying (Figure 6.1). They should be
characterized by the phenol-sulfuric acid method (Dubois et al.,
1956), with an expected content of ~47% reducing sugars and
~5% protein as determined by the Bradford assay.

–43–
The cytotoxic activity of the microalgal-enriched extract and particulate yeast β-glucan Yestimun®
[(1,3)-(1,6)--glucan] 85% pure from brewers' yeast (Quimivita) used as a control (ranging from 0.001
to 1% w/v final concentration), has to be tested. The extracts and Yestimun® cytotoxicity is assayed
using primary human dermal cell cultures NHDF using an MTT assay. Cell survival is also assessed
(see the Algaecom handbook for method details).
6.2.2 Fish testing
The experiment is designed to test the effect of the chrysolaminarin-enriched microalgae extract and
is compared to Yestimun® as the control (experimental design in Figure 6.2).
-Distribute the animals (n = 70) between eight cylindrical tanks (1 m2 surface) in an open flow-through
circuit. The animals are acclimated for one week before starting the experiment and are fed with
commercial diets (Skretting; 1% biomass).
-The microalgal-enriched extract (MA) is intraperitoneally (i.p.) injected using coconut oil (Renuka Agri
Organics LTD) as a vehicle to slow down and prolong the release of the chrysolaminarin-enriched
microalgae extract. For administration of the treatment the freeze-dried microalgae extract should be
suspended in PBS, then added to the same volume (1:1) of coconut oil and vortexed to generate an
emulsion. A control solution should be prepared by mixing PBS:coconut oil in a proportion of 1:1.

Microalgal‐Extract group MA_MA group

MA_C group

19.3 ± 3.3 g
Reinjection

Control group Control_Control group

Figure 6.2 Experimental design for testing the administration of chrysolaminarin-enriched microalgae extract. Sole
(19.3 ± 3.3 g) should be acclimated to the system for 1 – 3 weeks before i.p. injection. Microalgae-enriched extract
suspended in a coconut oil should be injected in the microalgae group (1mg/fish) and the sham control group should
receive the PBS emulsion (1:1). Injections can be repeated as many times as desired at weekly intervals. Mortality
should be monitored, and samplings can be carried out as desired but to capture different phases of the immune
response at 2 and 5 dpi.

-Enough biological replicates should be used to ensure robust analysis of the response. In the
Algae4a-b project four tanks (10 specimens each) were given i.p. injections (1 mg/fish, vol 100 µL)
and 3 control tanks were (10 specimens each) injected i.p. with the control solution (sham control
group). Mortality should be monitored daily.
-Analysis of the results of treatment should be monitored by sampling 2 and 5 days post-injection (dpi)
and the organs collected (n = 6 specimens/treatment).
-Animals should be euthanized using an overdose of phenoxyethanol (250 ppm final concentration),
and for analysis of the immune response the kidney, spleen and intestine should be collected.
-Samples for gene expression analysis should be fixed in RNAlater (Invitrogen) and stored at -80°C
until use.
6.2.3 Cumulative mortality
This is determined by measuring daily the number of dead fish. An example of the outcome of the
experiments conducted in Algae4a-b is given in Figure 6.3. In the graph the number of animals that

–44–
die should be monitored daily and the cumulative mortality determined daily by summing the dead fish
on successive days.

100 MM‐MA/MA‐C

Reinjection
C‐C

Cumulated Mortality
80

60

40

20

0
0 1 2 3 4 5 6 7 8 9 10 11 12 13
Days post‐injection

Figure 6.3 In vivo cumulative mortality in sole i.p. injected with microalgal-enriched extracts. The control group (c-c) and the
experimental groups MA_MA, MA_C are reported.

6.2.4. Expression profiles in immunological organs of sole treated with chrysolaminarin-enriched

microalgae extract
RNA extraction is performed as described in chapter 5. The immunomodulatory activity of microalgae-
enriched extracts can be assessed by measuring the change in gene transcript abundance of genes
related to the inflammatory response, cellular stress, carbohydrate binding receptors and defence
against bacteria and viruses. Relevant immunological organs include the kidney, spleen and intestine.
Typical gene transcripts used to assess the immune response are indicated in Table 6.1; the
responsiveness of the genes to i.p. injection of chrysolaminarin-enriched microalgae extract is also
noted.

Table 6.1 Expression data of the kidney, spleen and gut (after i.p. injection of the MA-enriched extract of a subset of 10 genes
with different functions (left). ns indicates if not differentially expressed. The arrow indicates if the gene was up- or down-
regulated and the sampling day.

Kidney spleen gut

injection reinjection injection injection

Proinflammatory cytokine il1b 2d ns 2d 2d

Proinflammatory cytokine tnfa ns ns ns ns

Cellular stress hsp90 2d 2d 2d ns

Complement C3 c3 ns ns 2d 5d

Antimicrobial peptide hamp1 2d ns 2d 2d

Sugar receptor clep 2d ns ns ns

Chemokine cxc10 ns ns 5d ns

Viral defense irf3 5d 2d 5d 2d

Bacterial defense lysg ns

The β-glucans from different sources are enhancers of non-specific immunity in fish through the
activation of macrophages and the production of pro-inflammatory cytokines. Intraperitoneal injection
in sole of microalgae-enriched extracts elicits a similar response to laminarin in other fish and rapidly
activates the expression of the pro-inflammatory cytokine il1b (at 2 dpi) in the kidney, spleen and
intestine. This cytokine acts as a mediator of β-glucan actions and triggers a generalized downstream
response through the NF-κB and MAPK signalling pathways to produce cytokines and activate the
migration and phagocytic activities of macrophages (Zou and Secombes, 2016). The spleen is a
major target for β-glucan actions with larger and more intense response than in other tissues (Douxfils

–45–
et al., 2017; Fredriksen et al., 2011; Lovoll et al., 2007). A previous study indicated that the activation
of tnfa was delayed with respect to il1b after β-glucan treatments in trout head kidney leukocytes
(Chettri et al., 200) and also showed dose-dependent effects as demonstrated by in vitro and in vivo
trials and an organ-specific response (Douxfils et al., 2017; Fredriksen et al., 2011; Lovoll et al., 2007;
Yuan et al., 2008).

6.3 Administration of microalgal extracts by oral intubation

To test the effects of yeast -glucans and microalgal extracts (MAe) on the Senegalese sole, extracts
in solution (prepared as described in section 6.2) are directly delivered to the intestine using an oral
intubation method. To optimize this methodology and establish the timeframe of uptake, some
preliminary trials were carried out.
-Intubation can be performed using a flexible polypropylene catheter 1.3 mm in diameter, attached to
a 1 ml syringe. The catheter should be introduced into the mouth from the blind side of the sole. All
the procedures should be carried out using anesthetized fish (150 ppm phenoxyethanol).
-To monitor the timeframe of uptake and dispersal after intra-intestinal delivery of the microalgae
extract, phosphate-buffered saline (PBS) containing a blue food dye (1.25% v:v) can be used as a
tracer (Figure 6.4).

Figure 6.4 Methods optimized to deliver extracts orally to directly expose the intestine. The pictures from left-right and top-
bottom indicate the procedure from extract preparation, cannulation and dissection. A blue food dye is used as a tracer to
establish the timing of gut transit. The last picture shows a negative control and a positive individual that absorbed the dye.

-Experiments of intubation in sole can be established once the intubation methodology is operational.
A typical experimental set-up is indicated in Figure 6.5. The experimental specimens are randomly
distributed between rectangular tanks (500 L, triplicate tanks/ treatment) in an open circuit at 21 ±
1°C, oxygen saturation >90% and salinity 42 ppt. Animals can be adapted for 1-3 weeks to the
experimental circuit prior to experiments using standard feeding regimes (e.g. commercial pellets,
Skretting at 2% tank biomass). Specimens of sole (or any other fish) should be fasted for two days

Oral intubation into the anterior intestine: Figure 6.5 An outline of the strategy
Solea senegalensis used in Algae4a-b to evaluate the
C ‐ Control ‐ PBS 1x
19.8 ± 0.52 g action of the microalgal extracts.
MA ‐ microalgal polysaccharide‐enriched extracts (PEE) Soles were intubated and provided
from P. tricornutum, abundant in chrysolaminarin β‐glucan
with four different solutions with the
Y – yeast β‐glucans (commercial) carrier control, MAe, yeast β-glucans
n=5‐12/group
Ph – whole Phaeodactylum tricornutum microalgae and whole microalgae (MA). Fish
were sampled short-term (3-48h) and
mid-term (7d) after introduction of the
Sampling after 3, 24, 48h: Sampling after 7d:
extracts and serum activities
Blood (n=5‐8) Enzyme activity assays Blood, Spleen, anterior gut (n=12)
analysed, gene expression or
Spleen (n=5‐8) Mid‐gut (n=12) DNA Microbiome lib.seq (pools n=5)
Anterior gut (n=5‐8)
RNA cDNA qPCR
qPCR confirmation
microbiomes. The logos indicate the
coordination between institutions.

–46–
before intubation to avoid uncontrolled effects of the intestinal contents.
Short-time responses of fish can be determined with intubation experiments by sampling at 3 h, 24 h
and 48 h. To determine the medium-term response sampling of fish at 7 days after oral intubation is
recommended. For sampling, fish should be anesthetized (200 ppm phenoxyethanol), and material
collected can include blood (to assess blood cells and enzyme activity in blood plasma) and tissues
(such as the spleen, anterior intestine and skin, collected into RNAlater, or fixed in ice-cold 4%
paraformaldehyde (PFA), pH 7.4 for histology). For microbiome analysis the intestine or skin is
recommended and 7 days after intubation should yield a mid-term response. Sample collection should
be carried out in a laminar flow cabinet, using sterilized dissection material and tissues should be
transferred to RNAlater, incubated at 4ºC for 24 h and then stored at -20ºC until DNA extraction for
the microbiome analysis.

6.4 Administration of microalgal extracts through the diets

The effects of microalgal extracts through the diet can be analysed using the following protocols.
6.4.1 Evaluation of crude extracts of P. tricornutum, yeast -glucans and encapsulated microalgal
extracts in fish juveniles
Specific diets for sole juveniles using microalgal bioactive extracts should be prepared in order to
better evaluate responses of the immune system in these animals that have fully developed
immunological organs. Diets should be formulated using a basal diet for sole (e.g. winflat, Krill meal,
squid meal, LT fish meal, wheat gluten, fish hydrolysate, gelatin, fish oil, lecithin) and microalgae
extracts can be included. In Algae4a-b four diets were prepared: Control diet without any additive;
Non-encapsulated chrysolaminarin (final concentration 0.1% w:w); encapsulated chrysolaminarin by
spray-drying (0.1%); Non-encapsulated particulate glucan from yeast (0.1%). The diets can be
supplied to soles for one month and growth should be monitored using a longitudinal approach (small,
fast growing juveniles are an ideal model). Mortality and growth should be determined (e.g. weight
and length) during the trial.
6.4.2 Evaluation of crude extracts of P. tricornutum and encapsulates in larvae throughout live prey
The efficiency of delivery of microalgal extracts to larvae can be assessed using live prey. Crude
extracts and encapsulates
using beta-cyclodextrin
(CD) or hydrolyzed Rice
A B C
Flour (HRF) are used to
enrich prey. Enrichment of
two prey can be assessed,
rotifers and artemia. The
delivery procedure is
shown in Figure 6.6. The
live prey should be
enriched for 30 min with
the microalgae extracts
and encapsulates by gentle D E F
rotation (Figure 6.6B). The
enrichment concentrations
can be varied in the
present trials: 2 mg/ml of
crude extracts in 2,000 live
preys /ml and 20 mg/ml for
encapsulates 10% in 2,000
Figure 6.6 Protocol for microalgal extract delivery using live preys. A) Liver preys were
live preys/ml were used. enriched for 24h with Tisochrysis and kept in the lab with gentle aeration. B) Incubation of
Three trials shouel be extracts with live preys; C) Delivery of larvae in plates to test the extracts; D) Delivery in
the wells; E) Plate structure; F) General overview of the trial.
carried out with the larvae
to robustly evaluate the

–47–
effectiveness of the oral delivery of the compounds. Assessments can include: a) administration of the
treatment through rotifers, b) dose effects of HRF encapsulates administered to the larvae through
rotifers and the effect of live prey filtering, and c) administration of the treatments through artemia.
The live preys should be supplied according to the normal feeding regime; in the sole, specimens are
supplied 10 prey/animal giving a final concentration of 2,000 rotifers/well or 200 artemia/well.
Mortality should be monitored for 48 h with full renewal of water every 24 h. The approach used in
Algae4a-b revealed that live prey is a very efficient way to deliver encapsulated and crude extracts to
sole larvae.
Samples of larvae can be taken for morphological, molecular and biochemical analysis (consult the
relevant section of the handbook for methods.

–48–
Chapter 7. Protocols for fish embryos and larval assays
7.1 Background knowledge
Young developmental stages of fish are extremely plastic and are useful to test bioactive compounds
for toxicity, epigenetic reprogramming or target effects on immunological and endocrinological
pathways. Although in aquaculture large tanks are generally required to test growth performance and
welfare parameters, the optimization of lab methods to screen potential actions is a valuable
mechanism to quickly screen molecules for specific action and predict their potential in the organism.
The use of microalgae as a source of a wide range of active molecules opens up a new research line
to bring together the biotechnology and the aquaculture sectors (Meena et al., 2013 Vetvicka et al.,
2013; Zekovic et al., 2005). These molecules can exert a plethora of actions with short- and long-term
responses that can be useful for aquaculture industry to improve their operational methods The
gilthead seabream (Sparus aurata) and Senegalese sole (Solea senegalensis) are two valuable fish
species mainly produced in Southern Europe but with markets worldwide (Cerda and Manchado,
2013; Manchado et al., 2016). These two species spawn for long periods releasing high amounts of
small eggs (<1 mm) that hatch in 24-48 h if incubated at 20ºC (Carballo et al., 2018b). Annually, an
excess of eggs is produced that can be used for a new biotechnological field, the "blue cosmetic"
based-industry, but also as a model for research of novel compounds to increase knowledge and
bring innovative solutions and efficient management to hatchery and nursery procedures. This is
particularly important in new aquaculture species such as sole, which demands major efforts to
provide new solutions to improve production parameters and promote industrial competitiveness.
In this section, valuable methods to work with young developmental stages of marine fish are
described. Moreover, some analytical methods are presented that will enhance the capacities to
evaluate the effects of novel compounds on growth, health and welfare.

7.1 Fish egg development and species selection

Gilthead seabream and Senegalese sole broodstocks of these species produce millions of eggs daily
since the breeders reproduce continuously during the whole spawning season (2-3 months as
average) offering embryos in a high number suitable for research in marine species. However,
spawns and egg characteristics are distinct between both species:
 In seabream, the spawns are activated in response to short photoperiod (8L:16D). This means
that the seabream releases its eggs in response to the switch on light conditions. Under captive
conditions, release is synchronized at switching on the lights. The eggs are characterized by a
central lipid droplet and 1 mm in diameter.
 In sole, the spawns are stimulated by thermoperiod and occur during the night requiring a
courtship behaviour.
 The sole eggs contain several small lipid droplets randomly distributed through the surface and
1 mm in diameter. The visualization of sole eggs is more difficult than seabream eggs due to
the colour of the egg, which is yellowish due to the pigments from the mussels provided to the
broodstock as feed (Figure 7.1).
Before proceeding to the evaluation of bioactive compounds, the following steps are necessary:
7.1.1 Evaluate the egg quality
- Separate the eggs by buoyancy in a metric cylinder and recover only the buoyant fraction.
- Estimate the fecundation rates (Total buoyant eggs/total eggs). As a rule, 1 ml contains
approximately 1,100 eggs. If fecundation rates are lower than 40%, discard the eggs for the trial.
- Distribute in a jar with aeration until use.

7.1.2 Monitor egg development

–49–
 To evaluate the effect of microalgae-based extracts on early developmental stages, the first step is to
establish the developmental stages in a species-specific since embryogenesis defines a time frame of
their sensitivity. As a general rule, early developmental stages are more sensitive to microalgae-
based treatments than older stages and can lead to high mortality rates. So, it is important to monitor
the embryonic stage before treatments in order to control later mortalities, correct development and
identify effects specifically linked to the treatments.
Previous studies in fish identified seven broad periods of embryogenesis as shown in Figure 7.1
(Kimmel et al., 1995):
 Zygote
 Cleavage
 Blastula
 Gastrula
 Segmentation
 Pharyngula
 Hatching

Morula blastula Gastrula Segmentation Pharyngula Hatched larvae

Figure 7.1 Developmental stages in gilthead seabream (upper panel) and sole (lower panel) embryogenesis

7.1.3 Criteria for species selection

-When early embryonic stages are required, the seabream is a good target species (Figure 7.1) since
the spawns occur in the morning when the lights are switched on.
-For longer exposure treatments, later embryonic stages (from gastrula onward) are required. In these
cases, sole is a good model since embryos and larvae are easily managed, they are at the final
stage of gastrula when collected from the tank (spawning is at midnight), larvae are easy to handle
and survival rates are very high.

7.2 Embryo and larval screening assays for bioactive compounds

7.2.1 Preparation of microalgae extracts for crude extracts
To prepare the extracts, the following steps are required:
-Microalgae homogenization. Microalgae biomass needs to be lyophilized and cells disrupted by high
pressure homogenization in cold buffer before preparing the microalgal extracts.
-Microalgae suspension.
-The dry mass is resuspended in seawater or propanediol. In the former, 1 g of microalgal
biomass is resuspended in 10 ml of filtered seawater and 0.01% DMSO added. The final

–50–
 volume of microalgae suspension will depend on the experimental design of the trial but
solutions should always be prepared in the same way and maintaining the ratio 1:10 w/v
constant. Once the solution is correctly hydrated and homogenized using a vortex or by
inverting the tube, it should be kept at 4ºC for at least 24 h before use.
-When propanediol is used, the microalgal biomass is resuspended in water:1,3 propanediol
(1:1) using the ratio 1:10 w/v and homogenized as described above. This solution should be
kept in the fridge at 4ºC for 7 days to avoid bacterial contamination.
- Extract preparation. Two hours before the exposure trial, this microalgae suspension is centrifuged
at 10,000 x g for 15 min at 4ºC and the soluble fraction collected in a new fresh tube and stored at
4ºC until use.
7.2.2 Preparation of materials for embryos and larval testing
-Preparation of water. Physicochemical and biological quality of the water is essential for a successful
trial irrespective of developmental stage.
-Water filtering. Hence, all water before use should be filtered through a 0.45 µm filter to reduce the
bacterial load. If the water contains large particles (>60 µm) or colloids, it should be pre-filtered
using a mesh of an adequate size and then filtered to remove the bacteria.
-Physicochemical control. Physicochemical parameters such as temperature, salinity and oxygen
should be controlled. The salinity should always be between 37-40 ppt and adjusted if necessary, to
ensure adequate buoyancy of the larvae. Moreover, the water should be adjusted to 20ºC to avoid
changes in the working temperature with a minimal oxygen concentration of 5 ppm.
-Water supplements. As microalgae represents a very rich medium for bacterial growth, antibiotic
solution (stock solution Penicillin-Streptomycin 100x; 10,000 U/ml) needs to be added to the water.
The antibiotics should be added just before use of the microalgae extract at a 1x final concentration.
Moreover, DMSO should be added at a final concentration of 0.01% in order to facilitate the
recovery of some polar compounds.
7.2.3. Plate selection and preparation
The plates used for fish trials should be customized to reduce larval manipulation and the application
of treatments. So, standard 12-well culture plates should be used and the well's bottom removed
using a heated metal wire (Figure 7.2). Special attention should be paid to fit the hole as much as
possible to the well diameter. After removal of the well base a 200 µm-mesh is glued to the plastic
well and sealed using silicone to avoid the loss of larvae during the assays.

Figure 7.2 Custom-made plates: Plate top (left panel) and bottom (right panel)

7.2.4 Procedure
-Washing. Plates must be washed and cleaned before use. Systematically, plates should be washed

–51–
 using a lab detergent to remove any organic material, rinsed in freshwater and finally rinsed in clean
distilled water to ensure the total removal of contaminating material.
-Sterilization (Figure 7.3). Plates are UV irradiated for 40 min to sterilize and avoid biological
contamination. After irradiation, a final volume of 150 ml of water medium should be added to each
tray.
-Assay preparation. Plates are placed in 500 ml disposable plastic trays (Figure 7.3).

Figure 7.3 Plates preparation before trials. UV irradiation (Left panel) and putting into their plastic trays (right
panel)

7.2.5 Embryo management

Transferring of embryos to the plates is a key step. The following steps are required (Figure 7.4):
-Concentration of embryos. To collect the high-quality embryo fraction from the total spawn, the
embryos should be transferred to a clean beaker in order to collect only the buoyant fraction (Figure
7.4A). This step can be carried out twice if required to ensure the collection of the best embryos
fraction. The same criteria are also applicable to larvae.
-Embryo delivery. To deliver 25-30 embryos per well, it is necessary to estimate the number of
buoyant embryos per ml in the top layer. Then, this volume can be taken using a plastic Pasteur
pipette with the tip cut off to avoid any physical damage to the eggs. If this is performed
systematically, the delivery of eggs tends to be uniform between wells. Although the optimal number
is around 30, up to 100 embryos can be maintained in the wells and still have similar hatching rates
(Figure 7.4B). Revise to check that the number is uniform in all the wells.
-Plates grouping. Once delivered the embryos/larvae between the wells of the multi-well plate, the
embryos are ready for incubation and treatments. Normally, the plates are stacked three-high and
gently agitated (50 rpm) at 22ºC in a lab incubator (Figure 7.4C).
-Water change. The water in the incubation trays should be changed daily or after the application of
the treatments. This can be done manually or using a vacuum pump as shown in Figure 7.4D. This
procedure is fast and does require manipulation of embryos or larvae. If the water needs to be fully
removed, the plate's tap can be slightly opened.

A B C D

–52–
 Figure 7.4 Embryos management. A) Buoyant fraction of high-quality embryos; B) Embryos in the well; C) Stacked plates
in the incubator; D) Removing water from the plates using a pump

7.3 Experimental design setting

For plate design:
-All experimental conditions should be
performed with triplicate plates and in
triplicate wells. All the wells within a
plate should be considered as a
technical replicate or pseudo-
replicates.
-The organization of plates within the
incubator should be optimized to
make management and data
collection as easier as possible. In Figure 7.5 Design of a 12-well plate: Six wells can be used for
general, within each plate there are survival estimation, three wells to collect larvae for gene
12 technical replicates, they can be expression analysis and three wells to measure the size of larvae
after microalgae treatments (left panel). Hatched larvae in the well
used for different purposes. For (right panel)
example: six-wells can be used for
survival estimates, three for biometric
measures, and three wells for gene
expression analysis (Figure 7.5).
-Plate labelling should be also in the plate cover side in such a way that the plate identification does
not interfere the embryo/larvae monitoring.

7.4 Protocol for microspheres delivery to larvae

7.4.1 Live prey enrichment
To deliver microencapsulates to larvae, they should be incorporated into live prey such as rotifers or
artemia. When administering microencapsulates via rotifers, 10 µm microspheres (as determined
using FluoSpheres Polystyrene) are not suitable from enrichment as concentrations from 1,000 to
3,000 spheres per ml in a
recipient containing 100
rotifers/ml are lethal in minutes
due to the big size of the particle
with respect to the rotifers mouth.
Smaller particles (from 1-2 µm)
are necessary for the proper
delivery of nutraceuticals to this
prey (Figure 7.6). In artemia,
concentrations of 10,000
spheres/ml for 100 artemia (100
spheres/artemia), result in rapid Figure 7.6 Delivery of FluoSpheres to live prey: Left panel: rotifers; Right
incorporation of the microspheres panel: artemia. The green balls are 10 µm spheres

–53–
into the gut without mortality in just 30 min (Figure 7.6).
7.4.2 Live prey delivery
-To deliver enriched artemia to sole larvae from 7 days post hatch (dph) onward, total artemia
metanauplii should be concentrated in a falcon tube (3-5 metanauplii/ml of tank water should be
provided).
-So, a concentrated stock (1,000x: i.e. 30,000 metanauplii/30 ml) has to be prepared.
-Then, half of the estimated spheres required to give a final concentration of 100 spheres/artemia are
added to the artemia stock and incubated for 15 min with slight agitation.
-Later, the remaining half of microspheres is added and incubated for other 15 min to ensure optimal
artemia enrichment.
-Thereafter, non-incorporated spheres are removed through a 350 µm mesh and artemia washed with
sterile seawater to remove all sphere residues. Artemia is recovered and delivered to the larvae
tanks to the desired concentration.
7.4.3 Monitoring
During the enrichment and delivery procedures the number of particles in the gut of artemia should be
constantly monitored under a microscope. Also, artemia and larvae should be fixed in formaldehyde
to estimate the degree of enrichment and delivery.

7.5 Survival analysis and samplings

To evaluate the effect of microalgae extracts on early developmental stages, survival can be
determined counting buoyant larvae and related to the total number of embryos or hatched larvae in
each well. This estimation should be done for at least six wells per plate and three plates per
treatment to calculate the mean ± standard deviation.
7.5.1 Biological parameters
To estimate the effects of microalgae treatments on length, live larvae from three-wells of each plate
are euthanized using MS-222 and fixed in 2 ml formaldehyde at 4%. Then, at least 25-30 larvae per
condition are photographed using a Leica DFC290 HD digital camera attached to a Leica DMIL LED
inverted microscope in a binocular (Nikon) using a scale and measured using ImageJ v1.47 software
(Figure 7.7).
7.5.2 Gene expression analysis
Live larvae from three wells of each plate are placed in
a new plate within a culture-filter (100 µm) within DEPC
water and washed twice and later transferred to an
Eppendorf tube containing 500 µl RNAlater and stored
at -80ºC until use.
7.5.3 Whole-mount in situ hybridization (WISH)
Larvae are anesthetized in MS-222 (0.125 mg/L) and
collected into ice-cold 4% PFA/1x PBS, pH 7, and kept
at 4 ºC overnight. Larvae are then washed in 1x PBS +
0.1% Tween-20 (Sigma-Aldrich) (PBT), bleached in 1x
PBS/0.5% KOH and 3% H2O2 at room temperature Figure 7.7 Larval measurement.
Larvae were measured using the
(RT) and transferred to 100% methanol (MeOH) and length from nose to the head-
stored at -20 ºC until use. vertebrate and to the tail. This
measure is always done using a
7.5.4 Histology calibrated image in ImageJ software.

Embryos and larvae are placed in 10 vol of ice-cold 4%

paraformaldehyde (PFA) pH 7.4 for overnight fixation at 4ºC.

–54–
7.5.5 Microbiome analysis
In microbiome samplings, protocols for fish aquaculture should consider the following points:
-All fish under comparison should share the same conditions in terms of water, tank, handling and
feeding.
-Water which the fish inhabit should always be collected as a baseline to discriminate authoctonous
from allochthonous microbes. The best way is to filter at least 2 l in triplicate through a 0.45 µm-filter
and then wash with RNAlater or a similar solution to preserve the sample.
-A representative sample of food should always be collected. Dry feed pellet or enrichment solutions
in larvae should be preserved following the manufacture’s recommendations. In the case of live
preys or microalgae, they are collected separately by filtering or centrifuging, and then fixed in
RNAlater or a similar solution for DNA analysis.
-During samplings
 All materials (tubes, scalpels, scissors) need to be autoclaved and sterilized prior to use.
Moreover, fish should be starved for at least 24 h unless the experimental design requires
them to be fed.
 Fish should be euthanized and sacrificed according to authorized procedures. If skin is
going to be sampled, a piece just on the top of the pectoral fin should be cut off and fixed
in paraformaldehyde for histological analysis (Figure 7.8).
 Skin should be carefully removed and placed in a petri dish to be fixed in RNAlater. Half of
the tissue is for gene expression analysis and the other for microbiome characterization.
 Special attention should be paid to the visceral cavity to avoid intestinal contamination. In
the case of gut, the cavity should be opened from genital pore using scissors with special
care to avoid opening the gut.
 Once the abdominal cavity is open, the intestine is cut just at the insertion in the liver
(anterior gut) and in the anus (posterior gut) and placed in a petri dish to remove
mesentery and fat.
 Then a segment of each region is cut off and fixed in RNAlater.
 As a general rule, all dissecting material should be flamed or cleaned with ethanol
between animals.

Examples for use and applications in Algae4a-b. Three exposure conditions were considered:
Short exposure embryos (SEE). Embryos were exposed to two concentrations of the microalgal
soluble fraction (ranging from 0.1 to 1 mg/ml) for 2 h. Moreover, an untreated control with no
extract was always included in parallel. After 2 h, all water was replaced as indicated and the
embryos incubated for a further 24 h to estimate hatching rates.
Long exposure embryos (LEE). Embryos were exposed to two concentrations of the microalgal
soluble fraction (from 0.1 to 1 mg/ml) for 24 h.
Short exposure larvae (SEL). Embryos were incubated in plates for 24 h until hatch. After removal
of dead embryos, the water was replaced by new water and two concentrations of the soluble
microalgae fraction added. At 2 h, the water was replaced by clean water and the survival rates
estimated after 48 h.

7.6 Larval testing

To test the biological performance of early developmental stages after exposure to microalgal
extracts, three challenges using heat, cold and salinity can be used to reveal differences in larval
response to these environmental stressors.
-Heat challenge.

–55–
  Water in the trays is replaced by preheated water at 28ºC.
 Then plates are placed in a heater at 37ºC for 40 min or when mortality in the control group
is close to 50%.
 Then, larval survival in at least 6 wells should be estimated by verifying the number of
animals with a heartbeat.
-Cold challenge.
 Water in the trays is replaced by precooled water at 4ºC.
 Then plates are located in a fridge at 4ºC for 2 h or when the mortality in the control group is
close to 50%.
 Then, larval survival in at least 6 wells should be estimated by verifying the number of
animals with a heartbeat.
-Salinity challenge.
 Water in the trays is replaced by distilled water.
 After 10 min at 20ºC without agitation, the water is again replaced by seawater and the
larvae incubated at 20ºC for 10 min.
 Then, larval survival is estimated by verifying the number of animals with a heartbeat.

7.7 Histological and Staining techniques

Histology and cytology permit a detailed evaluation by visual means and histological dyes of
developing immune tissues. In the case of barrier function, the structure of the skin, presence of
immune related cells, and mucous producing goblet cells permit potential changes in the barrier to be
detected. A number of different approaches based on staining using whole mount or paraffin
embedded and sectioned material can be used depending on the characteristics of the specimen.
7.7.1 Tissue processing and general histology
-Larval fixation and decalcification.
 Larval samples are fixed overnight at 4ºC in 4% PFA, pH 7.4 and then washed for 15 min in
three changes of sterile 1x PBS, followed by a final 15 min wash in Diethylpyrocarbonate
(DEPC) water.
 Then larvae are decalcified for 24 h in 0.5 M Ethylenediamine tetraacetic acid (EDTA) pH
8.0 in the dark and at room temperature.
 Decalcified larvae are washed in three changes of sterile DEPC water over 2 h.
-Cassette preparation and embedding.
 Specimens are transferred into histocassettes, processed and wax blocks generated.
 Specimens are dehydrated through a graded ethanol series (70%, 95% and 100%)
 They are cleared in xylene and impregnated with paraffin, using an automated tissue
processor (for instance, Leica TP1020).
 Finally, specimens are embedded in low melting point paraffin wax Histosec (58 ºC to 60 ºC;
Merck) using a Miles Scientific console (Figure 7.8). Alternative equipments can be used.

–56–
 Figure 7.8 Left image shows some typical paraffin blocks used for sectioning head and skin of larval sole. The right
image shows the rotary microtome and flotation bath used to generate sections and then mount them on slides,
respectively (photograph provided by Teresa Goncalves (MME-Ualg).

-Sectioning and quality control.

 Serial longitudinal sections of 5 μm of larvae are cut using a rotary microtome (for instance,
Leica RM 2135) and mounted on glass slides coated with 3-amino-propyltriethoxysilane
(APES)
 For staining, sections are dewaxed in two baths of xylene, 10 min each, and rehydrated
through a descending series of ethanol baths (100%, 95% and 70%) by immersing them
and gently agitating in each bath for 5 min before a final immersion (5 min) in distilled water,
just before staining.
 One section per individual is stained with hematoxylin and eosin to assess the general
quality of the sectioned tissue. Subsequently, 3 slides per individual containing 3 successive
sections per slide, with a gap of 4 to 5 sections (20 to 25 µm) between consecutive slides,
are stained with a combination of alcian blue-Periodic Acid Schiff (PAS)-Orange G for
histomorphometric analysis of melanomacrophages (MMCs) or granulocytes in the larvae,
or stained with Picro-Sirius Red for histomorphometric measurements of larval skin.
7.7.2 Haematoxylin and eosin staining
Haematoxylin and eosin staining (H&E) is used to stain respectively, the cell nuclei (blue/black) and to
distinguish the cytoplasm and connective tissue fibre/matrices (stained a variable shade of red,
orange and pink).
-After complete rehydration of sectioned
larvae, slides are immersed in Harris’s
haematoxylin for 5 min, following by bluing
in tap water for 5 min and rapidly rinsed in
distilled water (Figure 7.9).
-Slides are then immersed in eosin Y for 3
min, following an intensification of eosin
staining in distilled water with few drops of
acetic acid for 3 min.
-Final preparations are obtained by
Figure 7.9 Typical staining set-up with coplin jars
dehydration through a graded series of containing solvents and stains (photograph provided
ethanol baths (70%, 90% and 100%), by Teresa Goncalves).
followed by two clearing immersions in
xylene.
-Sections are mounted in DPX and a glass coverslip applied.

7.7.3 Combination of Alcian Blue, Periodic Acid-Schiff and Orange G of head kidney
The use of a combination of alcian blue/PAS and orange G permits neutral and acid mucins and also

–57–
 acidophils, basophils and chromophobes cells to be distinguished (Alcian blue stains acid mucins
deep blue and with the subsequent application of PAS stains the neutral mucins bright magenta,
while cells containing both acidic and neutral mucins appear purple.
-Alcian blue/PAS and orange G staining is performed on rehydrated larval sections by immersing
slides in 3% aqueous acetic acid for 1 min and then immersing them in 1% alcian blue for 1 h.
Excess alcian blue is removed by washing sections in tap water and then slides are immersed in 3%
aqueous acetic acid for 1 min, followed by a rinse in distilled water.
-Sections are oxidized with 1% periodic acid for 10 min, washed in running tap water for 5 min and
then immersed in Schiff reagent for 10 min.
-Sections are then washed in running tap water (5 min) and washed in 0.5% sodium methabisulphite
3 x 1 min each to remove excess fuchsin.
-Sections can be counterstained with Harris’s hematoxylin for 5 min and then washed again for 5 min
in running tap water, followed by a rinse in distilled water.
-A bleaching bath of 1% acid-alcohol for 3 min is followed by a wash in running tap water for 20 min
before staining in 1% orange G for 10 s and washing in distilled water until sections appear pale
yellow.
-Definitive preparations are obtained by dehydrating and clearing them in ethanol and xylene as
described above.
7.7.4 Picro-Sirius Red of skin
Picro-Sirius Red is a specific and sensitive stain of collagen fibres in vertebrate tissues, because the
acidic dye Sirius Red reacts strongly with the numerous basic amino acids of collagen fibres. This dye
also enhances birefringence of collagen fibres which is highly characteristic of collagen. In bright field
microscopy, collagen is red on a pale-yellow background. When visualized under polarized light,
larger collagen fibres are bright yellow or orange, while the thinner fibres (such as reticular fibres) are
green. Collagen type 4, which is a characteristic form of basement membranes and some types of
mucins also stain red with picro-sirius but are not birefringent.
-Completely dewaxed and rehydrated sections are stained in 0.1% Picro-Sirius Red for 1 h and then
washed in two changes of acidified 0.01 N HCl water for 1 min each.
-Definitive preparations are prepared by dehydrating and clearing them as described above.
7.7.5 Histomorphometric analysis
Stained larval sections are observed using a microscope (Leica DM2000) equipped with a digital
camera (Leica DFC480) and 1 section per slide photographed. The images obtained are analysed
using Fiji v1.47 software.

7.9 Whole-mount in situ hybridization (WISH)

To prepare samples for in situ hybridization analysis the following steps are required:
7.9.1 Probe preparation
-Amplification and cloning of target cDNA. PCR reactions to amplify target cDNA are done in a 25 μl
reaction volume containing:
 1 μl of cDNA template (synthetized from 100 ng of RNA)
 17.75 μl of sterilized distilled water
 2.5 μl of 10 mM dNTPs
 2.5 μl of 10x buffer
 1 µl MgCl2 (50 mM)
 0.5 μl of each of specific target primers (10 μM)
 0.25 μl (1.25 units) of Taq DNA polymerase

–58–
-The thermal cycle profile is as follows: 30 cycles of denaturation at 96°C for 30 s, annealing at 68°C
(annealing temperature will depend on the melting temperature of the primer pair designed for the
target gene) for 30 s, and extension at 72°C for 1 min.
7.9.2 PCR purification (using a vacuum protocol)
After checking the PCR product in agarose gel at 2%, the PCR reaction mix should be purified using a
PCR product purification kit and subsequently cloned into a TOPO vector and sequenced using
BigDye Terminator v3.1.
-Sample Preparation
 Add 400 μl of Binding Solution (D1) to the amplification reaction and mix thoroughly.
 Removal of oil is not necessary.
-Sample Loading
 Place the DNA Binding Plate on top of the vacuum manifold.
 Load the samples from previous step into the DNA Binding Plate (if the entire plate is not
used, we recommend covering the unused wells with aluminium plate sealers to maintain
the vacuum standard across all wells.
 Apply vacuum to the plate for 2 min.
-Plate Wash: Add 700 μl of Wash Buffer (D2) containing ethanol to each well of the plate.
-Apply vacuum for 2 minutes.
-Recover the sample in TE or water (50µl).

7.9.3 PCR Cloning

-Ligate the PCR product to a vector using T3 and T7 RNA polymerase recognition signals. Here we
describe the protocol using a TA vector for products amplified by PCR with a non-proof-reading
enzyme and with Topoisomerase (TOPO vectors). The ligation reaction contains 1 µl of salt solution,
0.5 µl of vector and 4 µl of Purified PCR (~5 ng) and should be incubated at RT for 15 min.
-Transform competent cells.
 Thaw a tube of chemical competent cells and add 2 µl of the ligation reaction.
 After incubating for 30 min on ice, give a heat shock of 42ºC for 30 s and then place the
tube on ice again and add 250 µl of SOC medium to the tube.
 Incubate the tube at 37ºC for 1 h with shaking (200 rpm).
 Then, spread 50 and 100 µl on LB plates containing ampicillin (50 μg/ml) and kanamycin
(50 μg/ml).
 Incubate at 37ºC overnight and check for the presence of recombinant colonies.
7.9.4 Sequencing of PCR products
-Mix the following reagents:
 6 µl of purified DNA template (3-10 ng) are added to 1 µl of reaction mix in BigDye®
Terminator v3.1 kit (Applied Biosystems).
 1 µl of 5X Sequencing Buffer in BigDye® Terminator v3.1 kit (Applied Biosystems).
 0.32 µl (3.2 pmol) of T3 or T7 primers (stock at 10 µM)
-Then, samples are incubated for 25 cycles at 96 C for 10 s, 52 C for 10 s, and 60 C for 3 min.

7.9.5 Riboprobe preparation

-RNA Probe synthesis (both sense and antisense probes should be prepared).
 Prepare the following reaction:

–59–
 - 2 μl of Buffer transcription 10x
- 2 μl dNTP-Dig RNA 10x Mix (kit Roche)
- 1 μl of T3/T7 Polymerase and 0.3 μl of RNAsin Promega (final volume 5.3 µl).
 Incubate at 37°C for 2 h in a thermal block.
 Then, add 2 μl DNase (RNase-free) and incubate at 37°C for 15 min. To stop the reaction,
add 1 μl EDTA 0.5 M pH 8 and 30 μl Milli-Q water.
-RNA Probe purification.
 Resuspend the column matrix (included in the kit mini Quick Spin RNA Columns for
purification of radiolabelled RNA) and open the cap of the column, remove the tip and put
the column into a 1.5 ml Eppendorf tube.
 Close the cap and centrifuge at 1,000 x g for 1 min at RT to remove any column buffer.
 Change the column to a clean tube and load the probe synthesis reaction (50 µl) and
centrifuge at 1,000 x g for 1 min at RT.
 Remove the columns and measure the concentration of the probe in a NanoDrop (2 µl).
 Also, prepare 2 µl of each probe and add 2 µl of formamide and 0.5 μl Blue Juice to load
onto an agarose gel. Once checked, dilute 1:1 in formamide and store at -20ºC.

7.9.6 Hybridization protocols

Steps to carry out the hybridization are the followings (Figure 7.11):
-Sample washing (in 2 ml Eppendorfs)
 75% MeOH/25% 1x PBS (37.5 ml MeOH 100% + 12.50 ml 1x PBS), 15 min
 50% MeOH/50% 1x PBS (25 ml MeOH 100% + 25 ml 1x PBS), 15 min
 25% MeOH/75% 1x PBS (12.5 ml MeOH 100% + 37.5 ml 1x PBS), 15 min
 PTW (1x PBS + 0.1% Tween 20% (5 ml 10x PBS + 44.75 ml DEPC water + 250 μl Tween
20%), 3 x 15 min
 1x PBS (5 ml 10x PBS + 45 ml DEPC water), 15 min.
 Cover with aluminium foil and store in the dark.
-Proteinase K treatment (in a multiwell plate):
 Proteinase K stocks have a concentration of 30 mg/ml (30,000 μg/ml). Prepare a working
solution of 10 μg/ml in 1x PBS, add 1 ml to each well, and then incubate at RT. The time for
proteinase K treatments depends on the larval size: 0-1 dph 10 min; 2 dph 14 min; 3-4-5-6-7
dph 16 min; 8-9 dph 20 min.
 Refixation in 4% PFA for 30 min.
 Four washes in PTW for 15 min (in orbital shaker).
-Preincubation in Hybridization Mix in the oven at 68ºC for 2 h.
-Store samples at -20ºC in Hybridization Mix solution if you do not intend to proceed with the
technique immediately.
-Preheat the samples stored in Hybridization Mix at 68ºC for 20 min.
-Incubate samples overnight at 68ºC and add 500 μl of Hybridization Mix + RNA Probe (50 ng probe-
Dig/200 μL Hyb Mix). For next steps, put into the oven the following solutions: Hyb(-); 75% Hyb(-
)/25% 2xSSC; 50% Hyb(-)/50% 2xSSC; 25% Hyb(- )/75% 2xSSC.
-Stringency washes (at 68ºC in 1 ml):
 Hyb(-) for 20 min
 75% Hyb(-)/25% 2xSSC for 20 min
 50% Hyb(-)/50% 2xSSC for 20 min
 25% Hyb(-)/75% 2xSSC for 20 min

–60–
  2x SSC/0.1% Tween for 20 min (5 ml 20x SSC + 250 μl Tween 20% + 44.75 ml DEPC
water)
 0.2x SSC/0.1% Tween 3 x 30 min (500 μl 20x SSC + 250 μl Tween 20% + 49.25 ml DEPC
water).
-Washing (at RT and in the shaker in 1 ml):
 75% 0.2x SSC Tween/25% MAB for 20 min (37.5 ml 0.2x SSC + 12.5 ml MAB)
 50% 0.2x SSC Tween/50% MAB for 20 min (25 ml 0.2x SSC + 25 ml MAB)
 25% 0.2x SSC Tween/75% MAB for 20 min (12.5 ml 0.2x SSC + 37.5 ml MAB)
 MAB for 20 min
 MABTr (MAB/0.1% Triton X-100 (Tr-X)) for 20 min (250 μl Tr-X 20% + 49.75 ml MAB).
-Preincubation at RT for 3-5 h (1 ml in the shaker): MAB/0.1% Tr-X/2% BS/10% Sheep Serum.
Incubation overnight at 4ºC (in 1.5 ml eppendorfs): MAB/0.1% Tr-X/2% BS/10% SS/α-DIG IgG
(1/5,000). The BS 5% is preheated in the thermoblock at 65 ºC before use.
-Washings (1 ml in the shaker): MAB-Tr-X 6 x 30 min (Dry the larvae carefully with paper in the final
wash).
-Developing (1 ml, cover in the shaker)
 Staining Buffer (0.1 M Tris-HCl pH=9.5/0.05 M MgCl2/0.1 M NaCl/0.1% Tween 20, 0.002 M
Levamisole), 3 x 15 min.
 Move the larvae to a microplate and add staining buffer (6.25 μl of NBT and 3.5 μl BCIP,
and 0.002 M of Levamisole). Observe the staining of the larvae after 15, 30 min or for as
long as is necessary until the desired staining intensity is acquired. If in 4 h the signal does
not appear or is very low renew this solution and put the microplate cover o/n at 4 ºC.
-Fixation in PFA 4% (1 ml, cover in the shaker)
 Fixation in PFA 4%, 20 min (1 ml).
 Washings in PTW, 4 x 15 min (1 ml).
-Storage (cover in a shaker)
 50% 1x PBS/50% Glycerol until the larvae sink to the bottom of the tube.
 20% 1x PBS/80% Glycerol until the larvae sink to the bottom of the tube.
 100% Glycerol o/n in a shaker, until the larvae sink to bottom of the tube and then store at -
20ºC.

A B C D

Figure 10.11 Example of WISH analysis. Positive signals are indicated in blue. A) dnmt1; B) dnmt3aa; C) dnmt3ab;
D) dnmt3b.See additional information in Firmino et al.2017

7.10 Enzymatic analysis of proteases

The hatching enzymes (or choriolysins) are proteolytic enzymes that digest egg envelope to release
fish embryos. These enzymes belong to the astacin metallo-protease family. They are expressed in a
transient group of cells called hatching gland cells that produce, accumulate, and secrete choriolysins
that will digest glycoproteins present on the internal side of the chorion, the internal zona radiata. Until

–61–
now, two main choriolysin types were reported, type I and II, encoded by several genes in the
genome. The type I (HCE-like) induces swelling of the egg envelope by cleaving the N-terminal region
and breaking the inter-molecular Glu-Lys cross-links. The type II participates in the solubilization of
the swollen egg envelope by cleavage at two specific sites. In addition to aquaculture, hatching
enzymes appear to be interesting as a target for cosmetics capable of delivering continuous
exfoliation without thinning or inflaming skin. The hatching enzyme is too large to penetrate beyond
the second layer of skin digesting dead skin cells but with no effect on living cells. Hence, we need an
adequate method to measure the levels of proteolytic activity after fish egg hatching.
7.10.1 Reagents
 2% (w/v) Azocasein Solution. Prepare stock in 1% Sodium bicarbonate (NaHCO3). Gentle
heating at 60ºC and stirring to form a solution (pH 8.3)
 10% (v/v) Trichloroacetic Acid (TCA) Solution (Prepare in deionized water using
Trichloroacetic Acid, 6.1 N Solution, 100% w/v)
 Buffer Tris 0.1 M pH 8.5 (Dissolve 12.1 g Tris base in 900 ml H2O, adjust to pH 8.5 with
concentrated HCl and then adjust volume to 1 l with deionized water)
 Sodium Hydroxide (NaOH) 2 M Solution (80 g in 1 l deionized water)
 Quick Start Bradford 1x Dye Reagent (Bio-Rad) or Bradford Solution for protein determination
(PanReac AppliChem)
 BSA standard (Quick Start Bovine Gamma Globulin Standard Set, Bio-Rad)
7.10.2 Procedures
-Egg collection
Some of the main challenges in choriolysin collection is to prevent the autodigestion due to its
proteolytic activity, to preserve the enzyme stability and to avoid contamination by egg by-products.
The eggs should be kept at concentrations ranging 100-200 egg/ml in sterilized clean marine
seawater and incubate at 20-22ºC until close to hatch. Then, water has to be carefully replaced by
clean water to collect the hatching enzymes. At hatching, the hatched larvae must be rapidly removed
to avoid an excessive contamination of hatching liquid. Thereafter, water is filtered to remove any
debris (40-100 µm mesh), aliquoted, and kept frozen -20ºC until use.
-Quantification of protein levels
 Prepare a BSA serial dilution or use the BSA standard (Quick Start Bovine Gamma Globulin
Standard Set, Bio-Rad). The linear range of these assays for BSA is 125–1500 μg/ml. For the
diluent, use the same buffer as in the samples. Protein solutions are normally assayed in
duplicates or triplicates.
 Take the 1x dye reagent from the 4°C storage and let it warm to ambient temperature. Invert
the 1x dye reagent a few times before use.
 Pipet 5 µl of each standard and unknown sample solution into microplate wells. Add 245 µl of
1x dye reagent to microplates wells and mix thoroughly.
 Incubate at room temperature for at least 5 min with shaking (60 rpm) in the dark. Samples
should not be incubated longer than 1 h at room temperature.
 Measure the absorbance at 595 nm of the standards, blanks, and unknown samples, using a
microplate reader.
 If your sample has a low protein concentration and cannot be measured, you can modify the
volume of sample from 5 µl to 20 µl, adding Bradford Reagent to a final volume of 250 µl. Use
a standard curve keeping the same proportion.
 Data analysis. If the spectrophotometer or microplate reader was not zeroed with the blank,
then average the blank values and subtract that value from the standard and unknown
sample values. Create a standard curve by plotting the 595 nm values (y-axis) versus their
concentration in μg/ml (x-axis). Determine the unknown sample concentration using the
standard curve. Adjust the final concentration by multiplying by the dilution factor used.
-Protease assay
The reaction is based in the following principle

–62–
 Protease (Conditions: Tª = 37°C, pH 8.5)
Azocasein + H2O -------------------> Coloured brownish Products
 Pipette (in µl) the following reagents into Eppendorf tubes correctly labelled:
Reagent samples
Blank sample1 sample2 sample3
Azocasein 250 250 250 250
Tris 100 100 100 100
Samples 0 300 300 300

 Incubate at 37ºC for 30 min

 Stop the reaction by adding 600 µl TCA in each tube (final volume = 650 + 600 = 1250 µl). In
the control add 300 µl of any sample. Then incubate 10 min on ice.
 Centrifuge at 13,000 rpm for 10 min
 Take 500 µl of supernatant and dispense into a new tube, and then add 500 µl of NaOH 2 M.
Mix by swirling and then, transfer a volume of 250 µl to a 96-well plate to measure the
absorbance at 440 nm.
 Calculation: ∆A440 nm = A440 nm Sample - A440 nm Blank.
You need to normalize the absorbance to 1 cm Light path. Try to use the automatic
correction in the spectrophotometer to set the pathlength to 1 cm if you use 96-well
plates. If not automatic, you need to normalize the absorbance value using the
following formula: OD 1 cm = OD sample/h (cm), in which h = height of liquid
sample filled in each well. To estimate h value, use the cylinder geometry of the wells.
For instance, with employed Nunc plates with a well diameter (d) of 6.5 mm, we can
estimate the height in the cylinder using the formula h = 4 x Vol/π x d2. Thus, if the
volume is 250 µl (mm3) in our measurement, then h = 4 x 250/3.14 x 6.52 = 7.53 mm.
In this way, to get the normalized OD, we should divide the measured OD by 7.53.
UNIT DEFINITION: If you define 1 unit of enzyme activity as the amount of protein that
resulted in an increase of absorbance at 440 nm of 0.01 (under these assay
conditions), you should follow the below calculation:

V1 = Total volume of assay (in ml), which is 1.25

df = Dilution factor
V3 = Volume (in ml) used in Colorimetric Determination, which is 0.125
V2 = Volume of enzyme used (in ml) which is 0.3
30 = time of incubation
* to determine the μmol of protein equivalent to ABS you will have to multiply the
OD by 100.
Units/ml=(OD440 x 100 x 1.25)/(30 x 0.3 x 0.125)

–63–
Chapter 8. in vitro dermal cell cultures and wound healing assays
8.1 Background knowledge
Cell culture is a process where isolated animal cells are maintained and grown outside of their bodies
under controlled conditions. In research they are used as alternative to animal experimentation to
investigate the effects of drugs and toxins in cell metabolism and survival (Baust et al., 2017). Isolated
cells can also be manipulated by transfection to investigate the role of genes in the physiology or
malignancy.
The skin is constantly exposed to many toxic environmental agents that can damage its structure and
integrity (Gallo, 2017). Two types of human skin cells, the keratinocytes and fibroblasts, that grow as
2D monocultures have been isolated and are commercially available, are mostly used in skin
research. In addition, human 3D (three dimensional)-skin models have also been developed and used
for in vitro toxicity testing, biomaterials evaluation, and investigation of fundamental biological
processes (Wells and Robinson, 2017). The most abundant cell type of the dermis are the fibroblasts
(Figure 8.1). These cells ensure skin firmness and elasticity and regulates collagen production. In
cosmetic industry research, fibroblasts are used to screen in vitro natural bio-compounds with
potential beneficial properties to increase skin heath. In skin research the scratch assay is a simple
and inexpensive method to study directional cell migration. This was the first and is still a vastly used
method to predict in vitro cell migration after skin damage provoked by the formation of a scratch
(”wound’’) in a monolayer of skin cells. In cosmetic research scratch assay is an in vitro method to
study the effect of natural compounds on the regeneration of cell-matrix and cell-cell interactions
during cell migration. In addition, skin suffers analogous processes to healing, and increasing the rate
of skin turnover has been proposed as a means to improve the appearance and quality of skin
(Ganceviciene et al., 2012).
Recently, some studies have highlighted the importance of the teleost skin as an alternative model for
dermatology as fish skin shares structural elements and biological processes with mammalian skin
(Figure 8.1). In addition, it has the advantage that it does not wrinkle or scar and the physiological
systems, including endocrine and immune system, tend to be less complex than in human. Fish
epidermis is made of living cells with a higher regenerative capacity and not by a layer of dead
cornified cells as in mammals (Rakers et al., 2010).

ep
se

sc
ep
dm

dm

msc

Figure 8.1 Structural characterization of a cross-section of the skin tissue from a fish (sea bream) and human. Fish
and human histological tissue sections were stained with Masson Trichrome. Ep: epidermis; sc: scale; se: squamous
epithelium; dm: dermis; msc: muscle

Here the manipulation of commercially available primary normal human dermal fibroblasts (NHDF)
(Figure 8.2) as well as primary teleost dermal fibroblasts (Figure 8.2) is outlined in the context of their
use as a routine assessment process for toxicity screening and the discovery of novel natural
microalgae extracts with application as cosmeceuticals.

–64–
 Figure 8.2 Human and zebrafish dermal fibroblasts. Left hand side: Normal human dermal fibroblasts (Lonza, USA)
at 100% confluence. The image was obtained with a Carl Zeiss microscope coupled with a Axiocam ERc 5s digital
camera. The characteristic flattened and extensible cell morphology of the fibroblasts is clear (amplification 4x). Right
® TM
hand side: Primary caudal fin fish fibroblasts SJD.1(ATCC CRL-2296 ) at 90% confluence. The image was obtained
with a Leica DM IL microscope coupled with a Visicam HDMI 6 digital camera (amplification 4x).

8.2 Culture of primary normal human dermal fibroblast cells

In this section, we describe the manipulation of a primary cell line isolated from normal human dermal
fibroblasts from adult. All media and reagents used are provided by LONZA for optimal cell growth,
however other less-rich medium can be used (e.g. DMEM) but cell growth is slower and changes in
fibroblast morphology can occur. Cryopreserved NHDF were purchase from the company and were
expanded and preserved according to the manufacturer’s directions. Cell stocks produced were
stored in the air phase of liquid nitrogen to ensure cell stability and viability. Here we provide the
general guidelines that are used for maintenance and growth of NHDF. All manipulations are
performed in a sterilized laminar flow for Class 2 Biosafety level. All the external surfaces of all
supplement vials and the medium bottles should be decontaminated with 70% ethanol upon use.
Before manipulation, growth medium and supplements should be warmed at 37ºC. NHDF cells are
grown at 37°C in a humidified 5% CO2 atmosphere.
8.2.1 Setting up a NHDF cell culture
-To set up a culture calculate the number of vessels needed based on the recommended seeding
density (3,500 cells/cm2) and the surface area of the vessel. Do not seed cells in well-plates directly
out of cryopreservation. Add the appropriate amount of medium to the vessels (1 ml/5 cm2) and
allow the vessels to equilibrate at 37°C, 5% CO2, in a humidified incubator for at least 30 min.
-Wipe the cell cryovial with 70% ethanol before opening. Under sterile conditions, twist the cap a
quarter turn to relieve pressure, then retighten.
-Quickly thaw the cryovial in a 37°C water bath being careful not to submerge the entire vial. Thawing
the cells for longer than 2 min results in less than optimal results.
-Resuspend the cells using a micropipette and dispense into the culture vessel. Gently rock the
culture vessel to evenly distribute the cells and return to the incubator.
-Τhe growth medium is changed the day after seeding and thereafter every other day. As cells
become confluent, the volume of medium increased: 1 ml per 5 cm2 at 25% confluence, 1.5 ml per 5
cm2 for 25 - 45% confluence and 2 ml per 5 cm2 over 45% confluence.

8.2.2 Cell subcultures

–65–
 The following instructions are for a 25 cm2 flasks. The volumes are adjusted accordingly for
other vessel sizes. Subculture the cells when they are 70 to 80% confluent.
-Before manipulation
 Thaw and warm at 37ºC: 2 ml of Trypsin/EDTA, 7-10 ml of HEPES Buffered Saline Solution
(HEPES-BSS), 4 ml of Trypsin Neutralizing Solution (TNS)
 Warm growth medium at 37ºC
 Prepare the new culture vessel. If more than one vessel is used subculture one at a time.
‐Cell manipulation
 Aspirate the medium from the grown culture.
 Rinse cells with 5 ml HEPES-BSS to remove medium remains.
 Aspirate the HEPES-BSS.
 Add 2 ml of Trypsin/EDTA (0.1 mM) solution and gently rotate the vessel to cover the cells
 Monitor cell detachment under a microscope.
 When 90% of the cells are rounded up (up to 2 to 6 min) tap the flask against the palm of your
hand to aid cell release. If only few cells detach wait 30 s to 1 min and tap again. If the
majority of cells do not detach within seven minutes, the trypsin is either not warm enough or
not active enough to release the cells.
 After cells detachment, neutralize trypsin with 4 ml Trypsin Neutralizing Solution.
 Transfer the detached cells to a sterile 15 ml centrifuge tube.
 Rinse the flask with a final 2 ml of HEPES-BSS to collect residual cells and add to the
centrifuge tube.
 Centrifuge the harvested cells at 220 x g for 5 min to pellet the cells. Aspirate most of the
supernatant.
 Flick the tube to loosen the pellet and resuspend cells in 2-3 ml of growth medium with the aid
of a pipette.
 Count cells and assess cell viability using a hemocytometer and Trypan Blue.
 Carefully transfer growth medium to new culture vessels by adding 1 ml growth medium for
every 5 cm2 surface area of the flask (1 ml/5 cm2). The recommended seeding density for
adult human Dermal Fibroblast Cells is 2,000-3,500 cells per cm2.
 Place the new culture vessels into a 37ºC, humidified incubator with 5% CO2.

8.3 Culture of fish dermal cells

The SJD.1 (ATCC® CRL-2296TM) primary cell line isolated from fibroblasts present in the caudal fin of
Danio rerio, is a highly used fish model in scientific experiments. The SJD.1 cells were acquired
cryopreserved and were expanded and preserved according to the manufacturer’s instructions. SJD.1
cells are cultured in complete Dulbecco’s modified Eagle’s medium (DMEM, Sigma, Spain) with 4.5 g/l
glucose, 110 mg/l sodium pyruvate and L-glutamine supplemented with 15% heat inactivated sterile
foetal bovine serum and 0.1% penicillin:streptomycin antibiotic mix (10,000 U:10 mg/ml, Sigma) and
250 μg/ml sterile filtered 1:100 amphotericin B solution (Sigma, Spain), in a humidified 10% CO2
incubator (Heraeus, Portugal) at 28ºC. Below we provide the general guidelines that are used for
maintenance and growth of SJD.1 cells. All the external surfaces of all supplement vials and the
medium bottles should be decontaminated with 70% ethanol before and after use, and before
manipulation, growth medium and supplements should be warmed at 28ºC. All manipulations are
performed in a sterilized laminar flow for Class 2 Biosafety level.
8.3.1 Setting up a SJD.1 cell culture
-To ensure the highest levels of viability of the cryopreserved cells, thaw and culture them
immediately upon arrival.
-Prior to thawing the cells, add normal culture medium into a 25 cm2 culture flask where the cells will
be maintained and allow it to equilibrate at 28°C with 10% CO2 in a humidified incubator for at least
30 min.

–66–
-Incubate the cryopreserved vial for 2-3 min in a 28ºC water bath with gentle agitation without
submerging the vials to prevent contaminations.
-As soon as the vial content is thawed remove it from the water bath and decontaminate by spraying
with 70% ethanol.
-From this step on all the procedure should be carried out under sterile conditions.
-Resuspend the cells using a micropipette and transfer them to a centrifuge tube containing 9.0 ml of
the complete culture medium and centrifuge at 125 x g for 7 min.
-Remove the medium and resuspend the cell pellet with complete culture medium and dispense into
the 25 cm2 culture flask previously prepared with gentle agitation to distribute the cells
homogenously.
-Incubate the culture flask with cells at 28ºC in an incubator with 10% CO2.
Culture medium should be changed every 2 to 3 days.

8.3.2 Cell subcultures

The cells should be subcultured when they reach 70 to 80% confluency. The volumes used in this
protocol are for a 25 cm2 culture flask; proportionally adjust the volumes accordingly for other type of
flasks.
-Before manipulation
 Before starting the subculture, process incubate the reagents needed at 28 ºC: 1x PBS, 0.25x
Triple Select supplemented with 0.53 mM EDTA (disruption reagent) and complete growth
medium.
-Cell manipulation
 Remove and discard the culture medium.
 Rinse the cell layer with 1x PBS to remove all traces of culture medium.
 Remove the 1x PBS and add 1 ml of TrypleSelect/EDTA to the flask for 5 to 10 min at 28 ºC,
and then observe cells under an inverted microscope to confirm that cells are detached and in
suspension.
 Add 3 to 4 ml of complete culture medium and resuspend the cells gently by pipetting up and
down.
 Add the desired volume of cell suspension to a new culture flask and gently stir for
homogenous dispersion. The recommended subculture rate is 1:3 to 1:4.
 Incubate the cells at 28ºC in an incubator with 10% CO2.

8.4 In vitro screening for skin bioactive compounds

Assessing the bioactivity of cosmetic products and ingredients used in cosmetic formulations is
essential to predict their effect on human skin. Several in vitro assays monitoring cell division and
proliferation, enzyme activity, membrane permeability, adherence, ATP production, co-enzyme
production and nucleotide uptake activity exist to elucidate and predict the effect of cosmetic products
in skin. Here we describe the cell cytotoxicity (MTT) assay which is the first assay for bioactivity
screening, and the scratch assay which is a targeted approach to study skin aging and repair.

8.4.1 MTT assay

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is a versatile and
popular assay for bioactivity screening. This colorimetric assay assesses cell metabolic activity as a
proxy for cell viability. In general, the MTT assay involves the conversion of the water soluble MTT to
an insoluble formazan. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which
reduce the MTT reagent to formazan. The formazan is then solubilized, and the concentration
determined by optical density at 570 nm. The darker the solution, the greater the number of viable,

–67–
metabolically active cells. Below the protocol of MTT Cell Proliferation Assay Kit (ThermoFisher
Scientific, USA) is described. The whole procedure can be performed in five hours (this does not
include cell preparation time). This method has the advantage of being easy, simple and applicable to
various cell-cell adhesion assays.
-Reagents preparation
 Prepare a 12 mM MTT stock solution by adding 1 ml of sterile 1x PBS to 5 mg of MTT. Mix by
vortex until dissolved. This will provide sufficient reagent for 100 tests, using 10 µL of the
stock solution per well. Stored for four weeks at 4°C protected from light.
 Add 10 ml of 0.01 M HCl to one tube containing 1 g of SDS. Mix gently by inversion until the
SDS is dissolved.
-Preparation of cells for the assay
 Plate cells at densities 5,000-15,000 cells per well of a 96 well-plate to reach 70-80%
confluence within 48-72 hours.
 The culture conditions can affect cell growth and results. The number of cell culture passages
for primary cultures (age of the cells), composition of the growth medium and
presence/absence of growth factors are important factors to have in consideration.
-Labelling cells with MTT
 For adherent cells, remove culture medium and replace it with 100 µl of fresh culture medium.
For non-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much
medium as possible and replace it with 100 µl of fresh medium.
 Add 10 µl of the 12 mM MTT stock solution to each well. Include a negative control of 10 µl of
the MTT stock solution to 100 µl of medium alone.
 Incubate at 37°C for 4 h to allow crystallization of the blue dye of formazan in the cells. For
high cell densities (>100,000 cells per well) the incubation time can be shortened to 2 h.
 Add 100 µl of the SDS-HCl solution to each well and mix thoroughly using the pipette to lyse
cells.
 Incubate the microplate at 37°C for 4 h using the humidified chamber. Longer incubations will
decrease the sensitivity of the assay.
 Mix and read absorbance at 570 nm.
8.4.2 MTT assay using primary teleost cells
MTT assay can also be applied for primary teleost fibroblasts to assess bioactivity of compounds. The
protocol is the same as described for primary human fibroblast with exception of the growing
conditions (28ºC with 10% CO2).
8.4.3 Scratch assay
The scratch assay is an in vitro assay to study directional cell migration mimicking the cell migration
process during wound healing in vivo. This is a simple and inexpensive method based on observation
of cell migration into a scratch (“wound”) created on the cell monolayer (Rodriguez et al 2005). In
cosmetic industry is used to target anti-aging agents since wound healing is related to skin
rejuvenation and repair. Interpretation of the data is dependent on cell passage and confluency.
Below there are examples of two assays to study cell: the cell migration assay (or barrier assay) and
the cell proliferation related (or scratch assay) assay. The main difference between these two assays
is the initial establishment of the cell scratch.
8.4.4 Cell migration assay (barrier assay)
The assay described is based on the CytoSelect™24 Wound Healing Assay kit (USA). The
CytoSelect™ 24-well plate Wound Healing Assay Kit contains treated plastic inserts (barrier) to create
a wound field with a defined gap of 0.9 mm to measure cell migration and proliferation (Figure 8.3).
With time, the migratory cells extend protrusions and invade and close the wound.
 Plate the appropriate number of cells to reach on the next day > 90% confluency.
 Remove the barrier.
 Add growth medium supplemented with the target compounds.

–68–
  Calculate the cell migration rate as: migration rate = length of cell migration (nm)/migration
time (h).

A B C

Figure 8.3 A) Wound healing assay plate, B) Wound field inserts; C) Wound filled surface area (images obtained from
cell biolabs).

8.4.5 Cell scratch assay

In this assay the “wound” is formed by scraping the cell monolayer in a straight line to create a
“scratch” using a regular p200 plastic pipette tip (Figure 8.4). Assays are normally performed in a six-
well plate or individual 6 mm cell culture dishes.
 Plate the appropriate number of cells to reach on the next day > 90% confluency.
 Generate a vertical scratch using a p200 plastic pipette tip.
 Remove the cell debris (to avoid cell deposition) with growth medium.
 Add 5 ml of medium supplemented with the target compound.
 To obtain the same field during the image acquisition, reference marks are created by etching
the dish lightly with a razor blade on the outer bottom of the dish or with an ultrafine tip
marker.
 Capture photographs using a phase-contrast microscope, leaving the reference mark outside
the capture image field but within the eye-piece field of view.
 Place the culture plate in the incubator at 37 °C for 8–18 h.
 The plate can be taken out of the incubator to be examined and photographed periodically
until complete coverages of the wound is reached. The time frame for incubation should be
determined empirically for the particular cell type used.

Figure 8.4 Scratch field of human fibroblasts with a regular p200 plastic pipette tip at different time points after scratching
(AS) (0 h, 6 h and 24 h). Image was obtained using a Carl Zeiss microscope that was coupled to Axiocam ERc 5s digital
camera (ampliation 4x).

–69–
8.4.6 Scratch assay on dermal fish fibroblasts
Scratch assay can be performed for dermal teleost fibroblasts as described before for human dermal
fibroblasts but changing the growing conditions (28 ºC with 10% CO2).

8.5. Isolation and primary culture of marine teleost fish dermal cells
The fish skin possesses a diversity of skin ornaments (scales scale-like and skin appendages) and is
covered with mucous as a consequence of their adaptation to the aquatic environments. Nonetheless
fish skin organization and structure is similar to humans and genes that regulate skin physiology and
skin repair and growth are common. Moreover fish skin has a higher potential to regenerate, the
epidermis is made of living keratinocytes and it does not scar or wrinkle, and the endocrine and
immune systems that regulate skin function are suggested to be less complex than in mammals
(Rakers et al., 2010). In addition fish skin or products derived from fish skin are currently been used to
stimulate human skin regeneration and recovery after burn traumas (Lima-Junior et al., 2019). Thus
fish skin or cells isolated from fish skin can represent alternative models to predict human skin
response and regulation to challenges with less ethical issues and lower costs associated with the
use of human skin or cells isolated from human skin. In this section we describe the protocol that was
developed to isolate marine fish keratinocytes and fibroblasts from skin and the optimized culture
conditions. The model species used was the European sea bass (D. labrax), one of the most popular
aquaculture teleost fish species produced in Europe. The origin of the tissues collected for the dermal
cultures are depicted in Figure 8.5. Attempts were also performed using the sole (S. senegalensis)
skin but the high content of protective skin mucous difficulted skin sterilization. Its association with a
large diversity of skin microbes was in part responsible for the short duration of the cultures as
contamination of those cultures was recurrent.

Scale
Caudal fin edge

Skin

Figure 8.5. Sea bass tissues from where dermal fish cells were isolated.

8.5.1 Culture media and conditions

The cultures are performed under a humid environment using an incubator with controlled
temperature in the absence of carbon dioxide. Cultures are maintained at 19-20ºC but a similar
performance is also observed at 27ºC. All manipulations are performed in a sterilized laminar flow for
Class 1 Biosafety level and sterile autoclaved material. All the plastics used are suitable for cell
culture. Composition of the culture media is: L15 (Sigma), 10% filtered Sea water, 10% heat
inactivated Fetal Bovine Serum (FBS, Sigma), 1 % penicillin:streptomycin antibiotic mix (10,000 U:10
mg/ml, Sigma), 250 μg /ml sterile filtered 1:100 amphotericin B solution (Sigma-Aldrich), and 2 mM L-
glutamine (Sigma). The employed seawater was collected from the fish natural environment (Ria
Formosa, Faro) and was sterilized by filtration with a 0.2 µM filter (Millipore).

–70–
8.5.2 Isolation of dermal cells from sea bass
Juvenile European sea bass of approximate 10-15 cm in length and weighting 30-50 g can be used.
Keratinocyte cultures can be also obtained using adult European sea bass (20-30 cm in length and
weighting 500-700 g). All fish used in this study were obtained from the Ramalhete Marine Station
(Centre of Marine Sciences, University of Algarve, Faro, Portugal) or from the EPPO - Estação Piloto
de Piscicultura de Olhão (IPMA - Instituto Português do Mar e da Atmosfera, Olhão, Portugal). Fish
are euthanized with 0.1% chemical euthanasia agent 2-phenoxyethanol solution diluted in natural
seawater followed by brain destruction. Animal manipulations must be performed in compliance with
international and national ethics guidelines for animal care and experimentation, under a “Group-I”
license from the Portuguese Government Central Veterinary service.

8.5.3 Skin-scales explants of juvenile sea bass

 Wipe the fish skin surface with 70% ethanol twice and with the aid of a tweezers pluck the
scales from below the dorsal fin.
 Wash the scales twice with sterile PBS supplemented with antibiotics for 5 min, and
subsequently 4 x 5 min each with fish cells culture media supplemented with antibiotics.
 Transfer 10-15 scales to a well of a 6-well plate and let them stand for 30 min to adhere to the
bottom. To increase tissue adherence, the wells of the 6-well plate can be previously coated
with inactivated FBS.
 Add 600-700 µl of culture media, enough to cover the scales (do not overflow; otherwise
scales will detach and no cell release will occur).
 Put the plate in the incubator and monitor the culture daily to check for potential
contaminations.
 After 2-3 days many cells will be released from the skin attached to the scale (Figure 8.6).
Most cells derived from scales are keratinocytes but some fibroblasts can also occur.
Fibroblasts will be attached to the plate while keratinocytes are normally in suspension.
Based on the adherent properties characteristic of the two different cell types, they can be
separated to obtain monocultures.

a b

kt
sc

kt

sc

Figure 8.6 Photographs of two distinct areas (a and b) of the culture plate of the sea bass skin-scale explant after three days in
culture. Keratinocytes are small and round in shape and they migrate out from the skin attached to the scale. Some clusters of
cell were also observed and may are in suspension. Images were obtained using a digital camera coupled to a Zeiss inverted
microscope. Magnification 20 x. sc- scale; kt- keratinocyte.

8.5.4 Skin explants of juvenile sea bass

 Wipe the fish skin surface with 70% ethanol twice and cut a 1 cm x 1cm slice of skin from
both fish flanks below the dorsal fin.
 Remove the muscle from the skin with the aid of a sterile scalpel.
 Cut the clean skin tissue in small pieces of ~1 mm in size.

–71–
  Wash the tissue fragments twice in sterile PBS supplemented with antibiotics for 5 minutes
and subsequently 4 x 5 minutes in fish cell culture media supplemented with antibiotics.
 Transfer 8-10 pieces of skin fragments to a well of a 6-well plate and let them stand for 30 min
to adhere to the bottom. To increase tissue adherence, the wells of the 6-well plate can be
previously coated with inactivated FBS.
 Add 600-700 µl of culture media, enough to cover the tissue. Do not overflow; otherwise cells
will not be released.
 Put the plate in the incubator and monitor the culture daily to check for potential
contaminations.
 After 2-3 days many cells will be released from the skin (Figure 8.7). Most cells derived from
the skin explants are fibroblasts. Nonetheless a mixed culture with keratinocytes can also be
obtained. Fibroblasts will attached to the plate while keratinocytes are in suspension. Based
on the adherent properties characteristic of the two different cell types, they can be separated
to obtain monocultures.

a b
fb

sk

fb

sk

Figure 8.7 Photographs of two distinct areas (a and b) of the culture plate of the skin explant after three days in culture.
Fibroblasts are the dominant cell type and they attached to the plate bottom. Images were obtained using a digital camera
coupled to a Zeiss inverted microscope. Magnification 20 x. sk- skin, fb- fibroblast.

8.5.5 Caudal edge fin explants of juvenile sea bass

 Wipe the fish skin surface with 70% ethanol twice and cut with a sterile scissors the edge of
the caudal fin.
 Wash the tissue 3 x 10 min in PBS supplemented with antibiotics and subsequently 3 x 10
min in culture medium supplemented with antibiotics.
 Cut with a blade the caudal edge in small pieces of ~1 mm in length.
 Transfer the fragments to the well of a 6-well plate (8-10 pieces per well) previously coated
with FBS. Let stand for 30 min to improve tissue adherence to the bottom well.
 Add 600-700 µl of culture media, enough to cover. Do not overflow; otherwise cell release will
not occur.
 Put the plate in the incubator and monitor the culture daily to check for potential
contaminations.
 After 2-3 days many cells will be released from skin (Figure 8.8). Many fibroblasts were
released from the fin explants. This is the main type of dermal cells isolated.

–72–
 a b
fb

fn
fb
fn

Figure 8.8 Photographs of the fin explant 3 days after culture from two distinct plate areas (a and b). Fibroblasts are the
dominant cell type and they attached to the plate bottom. Images were obtained using a digital camera coupled to a Zeiss
inverted microscope. Magnification 20 x. fn- fin, fb- fibroblast.

8.5.6 Isolation of skin keratinocyte from the scales of adult sea bass
 Wipe the fish skin surface with 70 % ethanol twice and remove the scales from below the
dorsal region of the two flanks with the help of a knife (back of the knife to avoid wounds in
the skin).
 Collect the scales in flacons (50 ml) with sterile PBS supplemented with antibiotics.
 Wash the scales (to remove the mucous) with agitation and discard the liquid. Repeat this
step for at least 3 times to ensure that the scales are clean.
 Add 5 ml of 0.25% trypsin/EDTA to each falcon and incubate for 1 h at room temperature with
agitation to facilitate cell release.
 Collect the supernatant and add 5 ml of PBS with antibiotics to the scales.
 Vortex scales for 1 min and centrifuge at RT for 5 min at 1,000 rpm. Collect supernatant.
 Resuspend scales (vortex) and centrifuge as described above for 3 times to increase cell
detachment.
 Centrifuge the collected cells at room temperature for 10 min at 1,000 rpm and discard the
supernatant.
 Repeat previous step twice.
 Resuspend cells in 5 ml of fish cell culture medium and vortex and centrifuge similarly as
described above. Repeat this step twice.
 After the third wash with media, resuspend the cell pellet in 5 ml of cell culture medium and
plate on a petri dish (10 cm diameter).
 Incubate the cells at 19º-20º C.
 On the following day skin keratinocytes (most dominant cell type, Figure 8.9) and skin
fibroblasts (attached to the plate) are released . Based on the adherent properties
characteristic of the two different cell types they can be separated to obtain monocultures.

Growth curve
6
a b

4
x 105

ktc

kt 0
0 50 100 150
hours –73–
Figure 8.9 a) Photograph of the sea bass keratinocytes culture isolated from adult skin-scales digested with trypsin after one
day of culture. Isolated keratinocytes as well as some keratinocytes cell clusters were observed. Image was obtained using a
digital camera coupled to a Zeiss inverted microscope. Magnification 10 x. b) Growth curve of the sea bass skin keratinocytes
primary culture during 110 hours after cell isolation. kt- keratinocyte, ktc- keratinocyte cluster.

–74–
Chapter 9. References
Anderson RA. Algal culturing techniques. Academia Press Publication, West Boothbay Harbor, 2005.
Anjo SI, Santa C, Manadas B. Short GeLC-SWATH: a fast and reliable quantitative approach for
proteomic screenings. Proteomics 2015; 15, 757-762
Anguís V, Cañavate JP. Spawning of captive Senegal sole (Solea senegalensis) under a naturally
fluctuating temperature regime. Aquaculture 2005; 243, 133-145.
Armengaud, J. Next-generation proteomics faces new challenges in environmental biotechnology.
Curr, Opin. Biotechnol. 2016; 38, 174-82.
Bayona KCD, Garcés LA. Effect of different media on exopolysaccharide and biomass production by
the green microalgae Botryococcus braunii. J. Appl. Phycol. 2014; 26, 2087-2095.
Basketter DA, Whittle E, Chamberlain M. Identification of irritation and corrosion hazards to skin: an
alternative strategy to animal testing. Food Chem. Toxicol. 1994; 32(6), 539-42.

Baust JM, Buehring GC, Campbell L, Elmore E, Harbell JW, Nims RW, Price P, Reid YA, Simione F.
Best practices in cell culture: an overview. In Vitro Cell Dev, Biol, Anim. 2017; 53(8), 669-672.
Benzekri H, Armesto P, Cousin X, Rovira M, Crespo D, Merlo M.A, Mazurais D, Bautista R, Guerrero-
Fernandez D, Fernandez-Pozo N, Ponce M, Infante C, Zambonino J.L, Nidelet S, Gut M,
Rebordinos L, Planas J.V, Begout M.L, Claros M.G, Manchado M, 2014. De novo assembly
characterization and functional annotation of Senegalese sole (Solea senegalensis) and
common sole (Solea solea) transcriptomes: integration in a database and design of a
microarray. BMC genomics 15, 952.
Benzie IFF, Strain JJ. The Ferric Reducing Ability of Plasma (FRAP) as a Measure of "Antioxidant
Power": The FRAP Assay. Analytical Biochem. 1996; 239(1), 70-6.
Bhunia B, Uday USP, Oinam G, Mondal A, Bandyopadhyay TK, Tiwari ON. Characterization, genetic
regulation and production of cyanobacterial exopolysaccharides and its applicability for heavy
metal removal. Carbohydr. Polym. 2018; 179, 228-243.
Bostock, J., McAndrew, B., Richards, R., Jauncey, K., Telfer, T., Lorenzen, K., Little, D., Ross, L.,
Handisyde, N., Gatward, I., Corner, R. Aquaculture: Global status and trends. Phil. Trans. R.
Soc. B 2010; 365, 2897–2912.
Botham PA1. The validation of in vitro methods for skin irritation. Toxicol. Lett. 2004;149(1-3), 387-90.
Bradford MM. A Rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72, 248-254
Caballero MA, Jallet D, Shi L, Rithner C, Zhang Y, Peers G. Quantification of chrysolaminarin from
the model diatom Phaeodactylum tricornutum. Algal Res. 2016; 20, 180-188.
Carballo C, Chronopoulou EG, Letsiou S, Maya C, Labrou NE, Infante C, Power DM, Manchado M.
Antioxidant capacity and immunomodulatory effects of a chrysolaminarin-enriched extracts in
Senegalese sole. Fish Shellfish Immunol. 2018a; 82, 1-8.
Carballo C, Firmino J, Anjos A, Santos S, Power DM, Manchado M. Short- and long-term effects on
growth and expression patterns in response to incubation temperatures in Senegalese sole,
Aquaculture 2018b; 495, 222-231.
Carballo C, Pinto PIS, Mateus AP, Berbel C, Guerreiro CC, Martinez-Blanch JF, Codoñer FM,
Mantecon L, Power DM, Manchado M. Yeast β-glucans and microalgal extracts modulate the
immune response and gut microbiome in Senegalese sole (Solea senegalensis). Fish
Shellfish Immunol. 2019; 92, 31-39.
Cerda J, Manchado M. Advances in genomics for flatfish aquaculture, Genes Nutr. 2013; 8(1), 5-17.
Chen CY, Yeh KL, Aisyah R, Lee DJ, Chang JS. Cultivation, photobioreactor design and harvesting of
microalgae for biodiesel production: a critical review. Bioresour. Technol. 2011; 102(1), 71-81.
Chen B, You W, Huang J, Yu Y, Chen W. Isolation and antioxidant property of the extracellular
polysaccharide from Rhodella reticulata. World J Microbiol Biotechnol 2010; 26, 833–840

–75–
Chettri JK, Raida MK, Holten-Andersen L, Kania PW, Buchmann K. PAMP induced expression of
immune relevant genes in head kidney leukocytes of rainbow trout (Oncorhynchus mykiss),
Dev Comp Immunol 2011: 35(4), 476-82.
Chew KW, Chia SR, Show PL, Yap YJ, Ling TC, Chang JS. Effects of water culture medium,
cultivation systems and growth modes for microalgae cultivation: A review. J. Taiwan Inst.
Chem. E. 2018; 91, 332-344.
Chiovitti A, Molino P, Crawford SA, Teng R, Spurck T, Wetherbee R. The glucans extracted with
warm water from diatoms are mainly derived from in- tracellular chysolaminaran and not
extracellular polysaccharides, Eur. J. Phycol. 2004; 39 (2) 117–128.
Codoñer FM, Ramírez-Bosca A, Climent E, Carrión-Gutierrez M, Guerrero M, Pérez-Orquín JM,
Horga de la Parte J, Genovés S, Ramón D, Navarro-López V, Chenoll E. Gut microbial
composition in patients with psoriasis. Sci. Rep. 2018; 8, 3812.
Daboussi F, Leduc S, Marechal A, Dubois G, Guyot V, Perez-Michaut C, Amato A, Falciatore A,
Juillerat A, Beurdeley M, Voytas DF, Cavarec L, Duchateau P. Genome engineering
empowers the diatom Phaeodactylum tricornutum for biotechnology. Nat. Commun. 2014; 5,
3831.
Dammak M, Hadrich B, Barkallah M, Hentati F, Hlima HB, Pichon C, Denis M, Fendri I, Michaud P,
Abdelkafi S. Modelling Tetraselmis sp. Growth-kinetics and optimizing bioactive-compound
production through environmental conditions. Biores. Technol. 2018; 249, 510-518.
Das P, Lei W, Aziz SS, Obbard JP. Enhanced algae growth in both phototrophic and mixotrophic
culture under blue light. Bioresour. Technol. 2011; 102, 3883-3887.
Delattre C, Pierre G, Laroche C, Michaud P. Production, extraction and characterization of microalgal
and cyanobacterial exopolysaccharides. Biotechnol. Adv. 2016; 34, 1159-1179.
De-Luca R, Trabuio M, Barolo M, Bezzo F. Microalgae growth optimization in open ponds with
uncertain weather data. Comp. Chem. Eng. 2018; 117, 410-419.
Douxfils J, Fierro-Castro C, Mandiki SN, Emile W, Tort L, Kestemont P, Dietary beta-glucans
differentially modulate immune and stress-related gene expression in lymphoid organs from
healthy and Aeromonas hydrophila-infected rainbow trout (Oncorhynchus mykiss), Fish
Shellfish Immunol 2017; 63, 285-296.
Draaisma RB, Wijffels RH, Slegers PM, Brentner LB, Barbols MJ. Food commodities from microalgae.
Curr. Opin. Biotechnol. 2013; 24(2), 169-177.
Dubois M., Gilles K.A., Hamilton J.K., Rebers P.A, Smith F. Colorimetric method for determination of
sugars and related substances. Anal Chem. 1956; 28, 350.
Egerton S, Culloty S, Whooley J, Stanton C, Ross RP. The gut microbiota of marine fish. Front.
Microbiol. 2018; 9, 873.
Falkowski PG, Katz ME, Knoll AH, Quigg A, Raven JA, Schofield O, Taylor FJ. The evolution of
modern eukaryotic phytoplankton. Science 2004; 305(5682), 354-360.
Firmino J, Carballo C, Armesto P, Campinho MA, Power DM, Manchado M. Phylogeny, expression
patterns and regulation of DNA Methyltransferases in early development of the flatfish, Solea
senegalensis. 2017; BMC Dev Biol 17, 11.
Fredriksen BN, Saevareid K, McAuley L, Lane ME, Bogwald J, Dalmo RA. Early immune responses in
Atlantic salmon (Salmo salar L.) after immunization with PLGA nanoparticles loaded with a
model antigen and beta-glucan, Vaccine 2011; 29(46), 8338-49.
Gallo RL. Human Skin Is the Largest Epithelial Surface for Interaction with Microbes. J. Invest.
Dermatol. 2017; 137(6): 1213-1214.
Ganceviciene R, Liakou AI, Theodoridis A, Makrantonaki E, Zouboulis, CC. Skin anti-aging strategies,
Dermatoendocrinol. 2012; 4(3): 308-319.
Gao Y, Lim TK, Lin Q, Li FY. Evaluation of sample extraction methods for proteomics analysis of
green algae Chlorella vulgaris. Electrophoresis 2016; 37, 1270–1276.
Garcia de la Serrana D, Estevez A, Andree K. and Johnston I. A. Fast skeletal muscle transcriptome

–76–
 of the gilthead sea bream (Sparus aurata) determined by next generation sequencing. BMC
Genomics 2012; 13 181.
George B, Pancha I, Desai C, Chokshi K, Paliwal C, Ghosh T, Mishra S. Effects of different media
composition, light intensity and photoperiod on morphology and physiology of freshwater
microalgae Ankistrodesmus falcatus – A potential strain for bio-fuel production. Biores.
Technol. 2014; 171, 367-374.
Gillet LC, Navarro P, Tate S, Röst H, Selevsek N, Reiter L, Bonner R, Aebersold R. Targeted data
extraction of the MS/MS spectra generated by data‐independent acquisition: a new concept
for consistent and accurate proteome analysis. Mol. Cell Proteom. 2012; 11, O111.016717.
Go S, Lee SJ, Jeong GT, Kim SK. Factor affecting the growth and the oil accumulation of marine
microalge, Tetraselmis suecica. Bioprocess Biosyst. Eng. 2012; 35, 145-150.
Goodrich JK, Di Rienzi SC, Poole AC, Koren O, Walters WA, Caporaso JG, Knight R, Ley RE.
Conducting a microbiome study. Cell. 2014; 158(2), 250–262.
Guermazi W, Masmoudi S, Boukhris S, Ayadi H. Under low irradiation, the light regime modifies
growth and metabolite production in various species of microalgae. J. Appl. Phycol. 2014; 26,
2283-2293.
Guillard RRL, Ryther RL. Studies of marine planktonicdiatoms. I Cyclotella nanahustedt and Denotula
confervalea (cleve) gran. Can. J. Microbiol. 1962; 8, 229-239.
Harper JW, Bennett JE. Proteome complexity and the forces that drive proteome imbalance. Nature
2016; 537(7620), 328-338.
Harris GW, Donovan BT. The pituitary gland: anterior pituitary, Univ of California Press, 1966.
Hoagland KD, Rosowski JR, Gretz MR, Roemer SC, Diatom extracellular polymeric substances -
function, fine-structure, chemistry, and physiology. J. Phycol. 1993, 29(5): 537-566.
Kamerling JP, Gerwig GJ, Vliegenthart JF, Clamp JR. Characterization by gas-liquid chromatography-
mass spectrometry and proton-magnetic-resonance spectroscopy of pertrimethylsilyl methyl
glycosides obtained in the methanolysis of glycoproteins and glycopeptides. Biochem. J.
1975; 151(3), 491-495.
Kawaroe M, Hwangbo J, Augustine D, Putra HA. Comparison of density, specific growth rate,
biomass weight, and doubling time of microalgae Nannochloropsis sp. Cultivated in Open
Raceway Pond Photobioreactor. AACL Bioflux. 2015; 8(5), 740-750.
Kim S.-K., Y. D. Ravichandran, S.B. Khan, and Y. T.Kim (2008) Prospective of the Cosmeceuticals
Derived from Marine Organisms. Biotechnology and Bioprocess Engineering 2008, 13: 511-
523.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. Stages of embryonic development of
the zebrafish, Dev Dyn. 1995; 203(3), 253-310.
Klindworth A, Pruesse E, Schweer T, Peplies J, Quast C, Horn M, Glöckner FO. Evaluation of general
16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based
diversity studies. Nucleic Acids Res. 2013; 41, e1.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 1970; 227(5259), 680-685.
Lawrence JN. Application of in vitro human skin models to dermal irritancy: a brief overview and future
prospects. Toxicol In Vitro. 1997 Jun;11(3):305-12.
Li L, Yan Y, Xu H, Qu T, Wang B. Selection of reference genes for gene expression studies in
ultraviolet B-irradiated human skin fibroblasts using quantitative real-time PCR. BMC Mol Biol.
2011; 12, 8.
Liang Y, Mai KS, Sun SC, Yu DZ. Effect of light intensity on the total lipid and fatty acid composition of
six strains of marine diatoms. Chin. J. Oceanol. Limnol. 2001; 19, 249-254.
Lima-Junior EM, de Moraes Filho MO, Costa BA, Fechine FV, de Moraes MEA, Silva-Junior FR,
Soares MFADN, Rocha MBS, Leontsinis CMP. Innovative treatment using tilapia skin as a
xenograft for partial thickness burns after a gunpowder explosion. J. Surg. Case Rep.
2019;2019(6):rjz181.

–77–
López-Elías JA, Esquer-Miranda E, Martínez-Porchas M, Garza-Aguirre MC, Rivas-Vega M, Huerta-
Aldaz N. The effect of inoculation time and inoculum concentration of the productive response
of Tetraselmis chuii (Butcher, 1958) mass cultured in F/2 and 2-F media. Arch. Biol. Sci.
2011; 63(3), 557-562.
Lovoll M, Fischer U, Mathisen GS, Bogwald J, Ototake M, Dalmo RA. The C3 subtypes are
differentially regulated after immunostimulation in rainbow trout, but head kidney
macrophages do not contribute to C3 transcription, Vet Immunol Immunopathol 2007: 117(3-
4), 284-95.

Manchado M, Planas JV, Cousin X, Rebordinos L, Claros MG. Current status in other finfish species:
Description of current genomic resources for the gilthead seabream (Sparus aurata) and
soles (Solea senegalensis and Solea solea), in: S.A. MacKenzie, S. Jentoft (Eds.), Genomics
in aquaculture, Elsevier, Cambridge, MA, USA, 2016, pp. 195-221.
Mandalam RK, Palsson B. Elemental balancing of biomass and medium composition enhances
growth capacity in high density Chlorella vulgaris cultures. Biotechnol. Bioeng. 1998; 59, 605-
611.
Martignoni M, Groothuis GM, de Kanter R. Species differences between mouse, rat, dog, monkey and
human CYP-mediated drug metabolism, inhibition and induction.Expert Opin. Drug Metab.
Toxicol. 2006;2(6):875-94.
Martyniuk CJ, Kroll KJ, Porak WF, Steward C, Grier HJ, Nancy D.Denslow ND. Seasonal relationship
between gonadotropin, growth hormone, and estrogen receptor mRNA expression in the
pituitary gland of largemouth bass. Gen. Comp. Endocrinol. 2009; 163, 306-317.
Meena DK, Das P, Kumar S, Mandal SC, Prusty AK, Singh SK, Akhtar MS, Behera BK, Kumar K, Pal
AK, Mukherjee SC. Beta-glucan: an ideal immunostimulant in aquaculture (a review). Fish
Physiol Biochem. 2013; 39(3), 431-457.
Meseck SL, Alix JH, Wikfors GH. Photoperiod and light intensity effects on growth and utilization of
nutrients by the aquaculture feed microalgae, Tetraselmis chuii (PLY429). Aquaculture 2005;
246, 393-404.
Montreuil J, Spik G, Chosson A, Segard E, Scheppler N. Methods of study of the structure of
glycoproteins. J. Pharm. Bel. 1963; 18, 529-546.
Montreuil, J., Bouquelet S., Debray H., Fournet, B., Spik G., Strecker G. Glycoproteines. In M. F.
Chaplin, & J. K. Kennedy (Eds.), Carbohydrates analysis: a practical approach. Oxford IRL
Press 1986; pp. 143-204.
Ogawa K., Ikeda Y., Kondo S. A new trisaccharide, α-d-glucopyranuronosyl-(1→3)-α-l-
rhamnopyranosyl-(1→2)-α-l-rhamnopyranose from Chlorella vulgaris, Carbohydrate Res,
1999: 321 (1–2) 128-131,
Perez-Garcia O, Escalante FM, de-Bashan LE, Bashan Y. Heterotrophic cultures of microalgae:
Metabolism and potential products. Water Res. 2010; 45(1), 11-36.
Pinto PIS, Guerreiro CC, Costa RA, Martinez-Blanch JF, Carballo C, Codoner FM, Manchado M,
Power DM. Understanding pseudo-albinism in sole (Solea senegalensis): a transcriptomics
and metagenomics approach. Sci. Rep. 2019; 9(1), 13604.
Phong WN, Show PL, Ling TC, Juan JC, Ng E-P, Chang J-S. Mild cell disruption methods for bio-
functional proteins recovery from microalgae - recent developments and future perspectives.
Algal Research 2018; 31, 506–516.
Ra CH, Kang CH, Jung JH, Jeong GT, Kim SK. Effects of light-emitting diodes (LEDs) on the
accumulation of lipid content using a two-phase culture process with three microalgae.
Bioresour. Technol. 2016; 212, 254-261.
Rakers S, Gebert M, Uppalapati S, Meyer W, Maderson P, Sell AF, Kruse C, Paus R., 'Fish matters':
the relevance of fish skin biology to investigative dermatology. Exp Dermatol. 2010 Apr; 19(4),
313-324.

–78–
Ranadheera, CS, Vidanarachchi, JK. 2013. Potentials and applicability of marine-derived
nutraceuticals in dairy industry. Agr. Food Ind. HiTech 24, 44-48.
Rizwan M, Mutjaba G, Memon SA, Lee K, Rashid N (2018). Exploring the potential of microalgae for
new biotechnology applications and beyond: A review. Renew. Sustain. Energy Rev. 2018;
92, 394-404.
Rodriguez LG, Wu X, Guan JL. Wound-healing assay. Methods Mol. Biol. 2005; 294, 23-29.

Safi C, Ursu AV,Laroche C, Zebib B, Merah O, Pontalier P-Y, Vaca-Garcia C. Aqueous extraction of
proteins from microalgae: effect of different cell disruption methods. Algal Res. 2014; 3, 61-
65.
Santa C, Anjo SI, Manadas B. Protein precipitation of diluted samples in SDS‐containing buffer with
acetone leads to higher protein recovery and reproducibility in comparison with TCA/acetone
approach. Proteomics 2016; 13, 1847-1851.
Sarropoulou E, Galindo-Villegas J, Garcia-Alcazar A, Kasapidis P, Mulero V. 2012 Characterization of
European sea bass transcripts by RNA-seq after oral vaccine against V. anguillarum. Mar
Biotechnol, 14, 634-642.
Schmid J, Sieber V, Rehm B. Bacterial exopolysaccharides: Biosynthesis pathways and engineering
strategies. Front. Microbiol. 2015; 6:496.
Schulze PSC, Pereira HGC, Santos TFC, Schueler L, Guerra R, Barreira LA, Perales JA, Varela JCS.
Effect of light quality supplied by light emitting diodes (LEDs) on growth and biochemical
profiles of Nannochloropsis oculata and Tetraselmis chuii. Algal Res. 2016; 16, 387-398.
Singh SP, Singh P. Effect of temperature and light on the growth of algae species: A review. Renew.
Sust. Energ. Rev. 2015; 50, 431-444.
Swettenham K. The buffered performic-acid-alcian-blue-periodic-acid-Schiff method for the
differentiation of basophils in the human and rat pituitary, J Clin. Pathol. 1960; 13(3), 256-260.
Suarez ER, Kralovec JA, Noseda MD, Ewart HS, Barrow CJ, Lumsden MD, Grindley TB. Isolation,
characterization and structural determination of a unique type of arabinogalactan from an
immunostimulatory extract of Chlorella pyrenoidosa. Carbohydrate Research 2005;
340,1489–1498
Tafalla C, Bogwald J, Dalmo RA, Adjuvants and immunostimulants in fish vaccines: current
knowledge and future perspectives, Fish Shellfish Immunol 2013; 35 1740-50.
Taha HM, Said HA, Abdel-aziz WM, Khaleafa AE. Effect of Zinc and Copper toxicity on growth and
some metabolites of the green alga Tetraselmis chuii Butcher. Egypt. J. Exp. Biol. (Bot.)
2012; 8(2), 183-192.
Tall A, Teillon A, Boisset C, Delesmont R, Touron-Bodilis A, Hervio-Heath D. Real-time PCR
optimization to identify environmental Vibrio spp. strains. J. Appl. Microbiol. 2012; 113, 361-
372.
Tine M, Kuhl H, Gagnaire P.A, Louro B, Desmarais E, Martins R.S, Hecht J, Knaust F, Belkhir K,
Klages S, Dieterich R, Stueber K, Piferrer F, Guinand B, Bierne N, Volckaert F.A, Bargelloni
L, Power D.M, Bonhomme F, Canario A.V, Reinhardt R. European sea bass genome and its
variation provide insights into adaptation to euryhalinity and speciation. Nature commun.
2014; 5 5770.
Tirichine L, Bowler C. Decoding algal genomes: tracing back the history of photosynthetic life on
Earth. Plant J. 2011; 66, 45-57.
Vadiveloo A, Moheimani NR, Cosgrove JJ, Bahri PA, Parlevliet D. Effect of different light spectra on
the growth and productivity of acclimated Nannochloropsis sp. (Eustigmatophyceae). Algal
Res. 2015; 8, 121-127.
Vetvicka V,Vannucci L, Sima P. The effects of beta-glucan on fish immunity, N. Am. J. Med. Sci.
2013; 5(10), 580-588.
Wahidin S, Idris A, Shaleh SRM. The influence of light intensity and photoperiod on the growth and
lipid content of microalgae Nannochloropsis sp. Biores. Technol. 2013; 129, 7-11.

–79–
Wells E, Robinson AS. Cellular engineering for therapeutic protein production: product quality, host
modification, and process improvement, Biotechnol J. 2017 Jan;12(1)
Yim JH, Son E, Pyo S, Lee, HK. Novel sulfated polysaccharide derived from red- tide microalga
Gyrodinium impudicum strain KG03 with immunostimulating activity in vivo. Marine
Biotechnology 2005; 7, 331-338
Yuan C, Pan X, Gong Y, Xia A, Wu G, Tang J, Han X. Effects of Astragalus polysaccharides (APS)
on the expression of immune response genes in head kidney, gill and spleen of the common
carp, Cyprinus carpio L, Int Immunopharmacol 2008: 8(1), 51-8.
Zekovic DB, Kwiatkowski S, Vrvic MM, Jakovljevic D, Moran CA. Natural and modified (1-->3)-beta-D-
glucans in health promotion and disease alleviation. Crit Rev Biotechnol. 2005; 25(4), 205-
230.
Zou J, Secombes CJ. The Function of Fish Cytokines, Biology 2016; (Basel) 5(2).

–80–