Recycling of the culture media for pilot scale production of

Arthrospira platensis (Spirulina)

Nádia Filipa Medronho Veiga

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisors:

Dr. Luís Filipe Amaro da Costa

Prof. Marília Clemente Velez Mateus

Examination Committee

Chairperson: Prof. Helena Maria Rodrigues Vasconcelos Pinheiro

Supervisor: Dr. Luís Filipe Amaro da Costa

Member of the committee: Prof. Frederico Castelo Alves Ferreira

October 2016

ii

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In memory of one of the great men of my life. Thank you for all.

iv

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Acknowledgements

First of all, I would like to express my sincere thanks and gratitude to all of those who made this

thesis possible and its preparation a constant learning.

To the board of A4F – Algae for future, who granted me the opportunity to perform a summer

internship and, consequently, to develop my master’s thesis in the company, increasing my

knowledge in the microalgae biotechnology area and allowing me to discover and work in a

laboratory and in a pilot production unit. I will never forget this unique experience.

To Luís Costa, my supervisor at A4F, for guiding me in my work and for helping me in the

development of this thesis and for always being available and committed to answer my questio ns

and discuss alternatives and different methodologies with me.

To Professor Marília Mateus for her advice during the course of this work.

To all members of A4F, specially my colleagues, for sharing their knowledge and helping me in the

worse moments; for sharing with me their bad and good days, including all the smiles and laughter,

songs and other crazy moments. In a general way, for making me feel at home.

I would like to thank LNEG (Laboratório Nacional de Energia e Geologia) for providing the sun-dryer

used in this project and for the daily radiation data.

To my parents, godparents and cousin for always loving and encouraging me and for their patience in

my bad days. Thank you for all the support that you gave me along all these years.

To my close friends who encourage me every day to strive towards my goals.

To my Sensei Rui Caipira for being a very important person in my childhood and teaching me the values

of hard-work and never give up.

To TFIST- Tuna Feminina do Instituto Superior Técnico for contributing to my growth, for being an

escape so many times and a hobby to release the stress of the quotidian. Especially to my board of

association for being patient with me so many times and for forgiving my absence in worse situations

when I was needed.

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Resumo

Neste trabalho objetivaram-se três temas relacionados com a cianobactéria Arthrospira platensis

(Spirulina) à escala piloto: comparação das produtividades em diferentes sistemas de cultivo;

recirculação do meio de cultivo e otimização da sua composição; e procura de meios alternativos para

o cultivo da Spirulina. Alguns aspetos secundários foram também explorados ao longo deste trabalho:

várias metodologias de secagem e respetivos impactos nas células.

Primeiramente determinou-se a produtividade de culturas de A. platensis ao longo do tempo em

sistemas de cultivo à escala piloto: um sistema localizado no interior de uma estufa e três sistemas

localizados no exterior. Nalguns sistemas foram efetuadas renovações com taxas entre os 30 e os 83%

que garantiram a permanência das culturas na fase linear de crescimento. Obtiveram-se para os

diversos sistemas produtividades areais entre os 3,8 e os 9,6 g/m2/dia.

Seguidamente analisou-se ao longo do tempo a produtividade no sistema de cultivo raceway

convencional com taxas de renovação e de recirculação de meio de 50-54% e 63-83%, respetivamente.

A composição elementar do meio de cultivo recirculado foi analisada com o objetivo de otimizar a

produtividade da cultura.

Concluiu-se que é possível cultivar Arthrospira platensis com recirculação do meio durante, pelo menos,

36 dias sem que ocorra perda de produtividade. Foi sugerido um ajuste à receita do meio nutritivo para

melhor combater as necessidades nutricionais demonstradas pela cultura.

Relativamente ao estudo de meios de cultura alternativos ficou comprovado que após adaptabilidade à

salinidade, é possível cultivar Spirulina num meio de cultivo salino.

Palavras-chave: Arthrospira platensis; Escala Piloto; Microalgas; Produtividade; Recirculação do meio

de cultivo; Spirulina

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Abstract

This work focused on three subjects related to Arthrospira platensis production: comparison of

productivity of different cultivation systems; medium recirculation and optimization of its composition;

and research for alternative cultivation media to cultivate Spirulina. Some secondary aspects were

explored throughout this project: several drying methodologies and their impact on cells.

Firstly the time course evolution of productivity of cultures of A. platensis in pilot-scale cultivation

systems was determined: one system located inside a greenhouse and three outdoor systems. In some

systems renewals with rates between 30 and 83% were performed, allowing the culture to remain in the

linear growth phase. For the various systems areal productivities between 3.8 and 9.6 g/m2/day were

obtained.

Secondly, the productivity of Spirulina culture in raceway with renewals rates between 50 and 54% and

medium recirculation rates between 63 and 83% was analyzed over time. The elemental composition

of recycled culture medium was analyzed with the objective to optimize the culture productivity.

It was concluded that Spirulina can be cultivated with medium recirculation for at least 36 days without

productivity loss. An adjustment to the nutritive medium recipe was suggested to better fit the nutritional

needs shown by the culture.

Concerning the study of alternative culture medium, it was proven that after salinity adaptability, it is

possible to cultivate Spirulina in a saline culture medium.

Key-words: Arthrospira platensis; Microalgae; Pilot scale; Productivity; Recirculation of culture medium;

Spirulina

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Contents

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

Resumo ......................................................................................................................... vii

Abstract .......................................................................................................................... ix

List of Tables ............................................................................................................... xiv

List of Figures .............................................................................................................. xv

List of Equations ........................................................................................................ xvii

List of Acronyms ...................................................................................................... xviii

Chapter 1: ...................................................................................................................... 1

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

1.1 Characterization of Arthrospira platensis ................................................................... 1 1.1.1 General characterization................................................................................................................... 1 1.1.2 Taxonomic characterization ............................................................................................................. 2 1.1.3 Morphologic characterization ........................................................................................................... 3 1.1.4 Biochemical composition .................................................................................................................. 4 1.1.5 Applications ........................................................................................................................................ 6

1.2 Cultivation Systems ....................................................................................................... 8 1.2.1 Flat-plate photobioreactor................................................................................................................. 8 1.2.2 Raceway pond (RW) ......................................................................................................................... 9 1.2.3 Cascade raceway (CRW) ............................................................................................................... 10 1.2.4 Tubular photobioreactor (PBR) ...................................................................................................... 11

1.3 Areal Productivity vs Volumetric Productivity ......................................................... 12

1.4 Culture Medium vs Nutritive Medium......................................................................... 13

1.5 Harvesting of Biomass ................................................................................................ 13 1.5.1 Centrifugation ................................................................................................................................... 14 1.5.2 Sedimentation .................................................................................................................................. 14 1.5.3 Filtration............................................................................................................................................. 15 1.5.4 Flocculation ....................................................................................................................................... 15

1.6 The Strategy of Recirculation of Culture Medium .................................................... 16

1.7 Drying of Biomass ........................................................................................................ 17 1.7.1 Spray-drying ..................................................................................................................................... 17 1.7.2 Freeze-drying ................................................................................................................................... 18 1.7.3 Drum-drying ...................................................................................................................................... 18 1.7.4 Sun-drying......................................................................................................................................... 19

Chapter 2: .................................................................................................................... 21

Framework and Goals ................................................................................................ 21

Chapter 3: .................................................................................................................... 23

xii

Materials and Methods ............................................................................................... 23

3.1 Reagents and Solutions .............................................................................................. 23

3.2 Equipment ..................................................................................................................... 24

3.3 Biological Material ........................................................................................................ 24

3.4 Cultivation Systems ..................................................................................................... 25

3.5 Culture Medium Formulation ...................................................................................... 30

3.6 Operational Procedures .............................................................................................. 33 3.6.1 Inoculation......................................................................................................................................... 33 3.6.2 Renewal ............................................................................................................................................ 33 3.6.2.1 Renewal without recirculation ..................................................................................................... 33 3.6.2.2 Renewal with direct recirculation................................................................................................ 34 3.6.3 Collecting culture medium for elemental analysis ....................................................................... 34 3.6.4 Collecting of biomass for biochemical analysis (protein analysis) ........................................... 34 3.6.5 Drying of biomass in spray-dryer vs drying of biomass in sun-dryer ....................................... 35

3.7 Analytical Methods ....................................................................................................... 37 3.7.1 Microscopic observation ................................................................................................................. 37 3.7.2 Determination of culture concentration ......................................................................................... 37 3.7.2.1 Determination of optical density (OD) ....................................................................................... 37 3.7.2.2 Dry weight (DW) ........................................................................................................................... 38 3.7.2.3 Packed cell volume (PCV) .......................................................................................................... 38 3.7.2.4 Correlations for determining culture concentration.................................................................. 39 3.7.3. Determination of the culture volumetric productivity .................................................................. 41 3.7.4 Determination of the culture areal productivity ............................................................................ 41 3.7.5 Determination of nitrate ion concentration ................................................................................... 41 3.7.6 Pigments analysis ............................................................................................................................ 42 3.7.7 Proteins analysis .............................................................................................................................. 43 3.7.8 Elemental analysis ........................................................................................................................... 43

Chapter 4: .................................................................................................................... 45

Results and Discussion ............................................................................................. 45

4.1 Assay 1: Comparison of productivity in different cultivation systems ................. 45 4.1.1 Productivity analysis of Arthrospira platensis in indoor flat-plate photobioreactor ................. 46 4.1.2 Productivity analysis of Arthrospira platensis in cascade raceway .......................................... 48 4.1.3 Productivity analysis of Arthrospira platensis in outdoor flat-plate photobioreactor .............. 50 4.1.4 Productivity analysis of Arthrospira platensis in conventional raceway ................................... 51 4.1.5 Comparison between productivities analysis............................................................................... 53

4.2 Assay 2: Analysis of a medium recirculation strategy of Arthrospira platensis

production at a pilot-scale in a conventional raceway .................................................. 58 4.2.1 Productivity analysis of Arthrospira platensis culture using recycled culture medium .......... 59 4.2.2 Elemental analysis of fresh and recycled culture medium......................................................... 65

4.3 Assay 3: Analysis of Arthrospira platensis growth in alternative culture medium

.............................................................................................................................................. 69

Chapter 5: .................................................................................................................... 71

Conclusions and Future Work .................................................................................. 71

xiii

Chapter 6: .................................................................................................................... 73

Bibliography ................................................................................................................ 73

xiv

List of Tables

Table 1: Accepted classification for Arthrospira platensis .............................................................................. 2

Table.2: Comparison between open ponds and closed photobioreactors ................................................. 12

Table 3: List of reagents used in this work and their respective supplier. ................................................. 23

Table 4: List of reagents used in this work and their respective supplier. ................................................. 24

Table 5: Equipment used in the project and its respective model and manufacturer. ............................. 24

Table 6: Summary of cultivation conditions of the all cultivation systems. ............................................... 29

Table 7: Composition of culture medium reference recipe: SAG medium. Modified from (Aiba & Ogawa,

1977)............................................................................................................................................................ 30

Table 7: Composition of culture medium reference recipe: SAG medium. Modified from (Aiba & Ogawa,

1977)............................................................................................................................................................ 31

Table 8: Comparison between the reference and recipe developed by A4F according to the optimization

assay performed. ....................................................................................................................................... 32

Table 9: Comparison between spray-dryer and sun-dryer ........................................................................... 36

Table 10: Summary of factors that have impact in productivity. Productivities of the different cultivation

systems under study. ................................................................................................................................ 54

Table 11: Comparison between average areal productivity obtained and the average areal productivity

present in literature .................................................................................................................................... 55

Table 12: Renewal cycles of conventional raceway ..................................................................................... 59

Table 13: Summary of some parameters that can influence the culture productivity of each renewal

cycle............................................................................................................................................................. 63

Table 14: Results of protein analysis of each renewal cycle of conventional raceway ........................... 64

Table 15: Percentage of concentration variation between fresh medium and recycled medium. Only

variations which were equal or superior to 30% in absolute value were considered relevant and

therefore specified. .................................................................................................................................... 65

Table 16: Suggestion of reformulation of nutritive medium put forward in Table 7.Comparison between

the reference recipe developed by A4F and the suggestion of nutritive medium according to the

assay performed. ....................................................................................................................................... 68

xv

List of Figures

Figure 1: Linear and spiral filaments of Arthrospira platensis (magnification of 40 x). .............................. 3

Figure 2: Linear filament in reproduction (magnification of 60 x). ................................................................. 3

Figure 3: Extraction of all pigments present in Arthrospira platensis. .......................................................... 6

Figure 4: Extraction and separation between chlorophylls (green phase) and zeaxanthin (orange phase).

........................................................................................................................................................................ 6

Figure 5: Extraction of phycocyanin present in Arthrospira platensis. ......................................................... 6

Figure 6: Women in Lake Chad working in sand filter to obtained “dihe”. ................................................... 7

Figure 7: Outdoor flat-plate photobioreactors. ................................................................................................. 8

Figure 8: Representative scheme of a raceway pond to cultivate microalgae and cyanobacteria. ......... 9

Figure 9: Cascade raceway set-up. ................................................................................................................. 10

Figure 10: PBR with horizontally displayed tubes. ........................................................................................ 11

Figure 11: Microalgae biomass harvested by centrifugation from a culture. ............................................. 14

Figure 12: Scheme of microalgae production process operating without recirculation of culture medium.

...................................................................................................................................................................... 16

Figure 13: Scheme of microalgae production process operating with recirculation of culture medium. 17

Figure 14: Aspect of Spirulina dried in spray-dyer. ....................................................................................... 18

Figure 15: Aspect of Spirulina dried in sun-dryer. ......................................................................................... 19

Figure 16: Flat-plate photobioreactor used and located inside of greenhouse (day 0- after inoculation).

...................................................................................................................................................................... 25

Figure 17: Cascade raceway used (day 0- after inoculation). ..................................................................... 26

Figure 18: Flat-plate photobioreactor used and located outdoor (day 0- after inoculation). ................... 26

Figure 19: Cascade raceway used after some adjustments (day 0- after inoculation). ........................... 27

Figure 20: Conventional raceway used (day 0- after inoculation). .............................................................. 28

Figure 21: Biomass obtained by PCV. ............................................................................................................ 38

Figure 22: Calibration curve between OD730 measured by the spectrophotometer and the DW measured

at 180ºC in the moisture analyser for Arthrospira platensis. ............................................................... 39

Figure 23: Calibration curve between OD730 measured by the spectrophotometer and the PCV obtained

by centrifugation for Arthrospira platensis. ............................................................................................ 39

Figure 24: Calibration curve between PCV obtained by centrifugation and the DW measured at 180ºC

in the moisture analyser for Arthrospira platensis. ............................................................................... 40

Figure 25: Aspect of the culture after addition of NaOH (1M), heating and centrifugation. .................... 43

Figure 26: Final aspect of the samples that were read in spectrophotometer. ......................................... 43

Figure 27: Daily DW and average daily radiation in indoor flat-plate photobioreactor throughout the

assay. Radiation values refer to outdoor, whereas the radiation impinging on the indoor PBR can

be 50-80% lower according to the time of the day and to the day of the year. ................................ 46

xvi

Figure 28: Daily volumetric productivity and average daily radiation in indoor flat-plate photobioreactor

throughout the assay. Radiation values refer to outdoor, whereas the radiation impinging on the

indoor PBR can be 50-80% lower according to the time of the day and to the day of the year. ... 47

Figure 29: Daily DW and average daily radiation in cascade raceway throughout the 2nd assay. ......... 48

Figure 30: Daily volumetric productivity and average daily radiation in cascade raceway throughout the

2nd assay. .................................................................................................................................................... 49

Figure 31: Daily DW and average daily radiation in outdoor flat-plate photobioreactor throughout the

assay. .......................................................................................................................................................... 50

Figure 32: Daily volumetric productivity and average daily radiation in outdoor flat-plate photobioreactor

throughout the assay. ............................................................................................................................... 50

Figure 33: Daily DW and average daily radiation in conventional raceway throughout the assay. ....... 51

Figure 34: Daily volumetric productivity and average daily radiation in conventional raceway throughout

the assay. .................................................................................................................................................... 52

Figure 35: Contaminant (pollen) observed by microscopic observation of a sample of conventional

raceway (magnification 40x). ................................................................................................................... 52

Figure 36: Daily DW and average daily radiation in conventional raceway throughout the assay. ....... 60

Figure 37: Daily volumetric productivity and average daily radiation in conventional raceway throughout

the assay. .................................................................................................................................................... 60

Figure 38: DW and daily volumetric productivity in conventional raceway throughout the assay. ......... 61

Figure 39: Average areal productivity in each renewal cycle in conventional raceway. .......................... 62

Figure 40: Average areal productivity per unit of incident radiation in each renewal cycle in conventional

raceway. ...................................................................................................................................................... 62

Figure 41: Comparison of the different factors that can influencing the culture productivity in each

renewal cycle. Average radiation in MJ/m2 and Specific growth rate in day-1. ................................. 63

Figure 42: Evolution of the optical density (630 nm) of Arthrospira platensis in a culture medium (control)

and in a culture medium with 5 g/L of sodium chloride. ....................................................................... 70

Figure 43: Evolution of the optical density (630 nm) of Arthrospira platensis in a culture medium (control)

and in a culture medium with 7.5 g/L of sodium chloride..................................................................... 70

xvii

List of Equations

Equation 1: Determination of dry weight ......................................................................................................... 38

Equation 2: Determination of culture volumetric productivity. ...................................................................... 41

Equation 3: Determination of culture areal productivity. ............................................................................... 41

Equation 4: Correction of determination of nitrate ion concentration. ........................................................ 42

Equation 5: Lambert-Beer Law. ....................................................................................................................... 42

xviii

List of Acronyms

Acronyms

Full name

A4F A4F – Algae for future

AP Areal productivity

CRW Cascade raceway

DW Dry weight

FACS Fluorescence-activated cell sorting

LNEG Laboratório Nacional de Energia e Geologia

MO Microscopic observation

OD Optical density

PBR (Tubular) photobioreactor

PCV Packed cell volume

RW Raceway (conventional)

VP Volumetric productivity

1

Chapter 1:

Introduction

1.1 Characterization of Arthrospira platensis

1.1.1 General characterization

Arthrospira platensis (Spirulina) is a multicellular, filamentous, photosynthetic, gram-negative and non-

toxic cyanobacterium so it has the capacity to do photosynthesis using sunlight and carbon dioxide as

energy and carbon sources, respectively, to produce carbohydrates and proteins and release oxygen

that was produced during the process (Belay, 2002; Charpy, José, & Alliod, 2008).

Spirulina can be also called a “blue-green alga”, based on the wavelengths of the light it is able to absorb

(reason for the prefix ‘cyano’). It is important not to mistake this microalga with the marine

cyanobacterium with the scientific name of Spirulina subsalsa (Jourdan, 2006).

Arthrospira platensis is also symbiotic and extremophile, more precisely basophile since growth can

occur at pH between 8.5 and 11.5. However, the best pH range for growth is between 9 and 10 (Charpy

et al., 2008; Jourdan, 2006). Therefore, Spirulina cells develop better in hot, alkaline waters, rich in

nutrients with phosphorous and nitrogen. However, it can also grow in waters with some salinity (Charpy

et al., 2008; Tietze, 2004). On the other hand, the risk of contaminations in cultures of Spirulina is lower

because there are few microorganisms that can grow in this pH range (Jourdan, 2006).

In relation to temperature, Spirulina grows well in a range of temperatures above 10 ºC and below 40

ºC, however the best temperature for growth is around 35ºC (Charpy et al., 2008; Jourdan, 2006;

Vonshak, 2002).

The uses and mass cultivation of this cyanobacterium have risen substantially due to an increased

understanding of its biological systems.

.

2

1.1.2 Taxonomic characterization

Arthrospira platensis is a cyanobacterium which corresponds to the scientific classification present on

Table 1 (Charpy et al., 2008).

Table 1: Accepted classification for Arthrospira platensis

Domain Bacteria

Kingdom Eubacteria

Phylum Cyanobacteria

Class Cyanophyceae

Order Oscillatoriales

Family Phormidiaceae

Genus Arthrospira

Species platensis

The current designation of Spirulina for species of the genus Arthrospira, especially for A. platensis,

holds a more traditional, technological and practical meaning than a taxonomic one. However this

designation often can create confusion. It is important to stress that the genus Arthrospira is different

and phylogenetically distant from Spirulina, although they share the same spiral shape (Vonshak, 2002).

Recently, more evidence based upon 16S rRNA sequence, gas vacuolated cells and fatty acid

composition revealed the difference between the genus Arthrospira and Spirulina, (Richmond, 2004).

Even though the generic name Arthrospira is accepted, throughout this dissertation the name Spirulina

platensis will be used.

3

1.1.3 Morphologic characterization

At a morphologic level, the cells of Spirulina can form filaments that can be linear (filaments constituted

by juxtaposed cells) or spiral having both dimensions of 100-250 µm (Figure 1). The filaments can have

10 to 12 µm of diameter and when in a spiral form, filaments have 6 or 7 spires. The common name of

this cyanobacterium derives precisely of its spiral form.

Figure 1: Linear and spiral filaments of Arthrospira platensis (magnification of 40 x).

Once a filament has converted to the linear form due to a mutation that affects the cells during certain

growth conditions, either physical or chemical treatments, for example by UV radiation, or in a natural

way, it does not revert back to its spiral form. In a culture with spiral filaments, if few filaments become

linear, they tend to become predominant (Eykelenburg, 1980; Vonshak, 2002; Wang & Zhao, 2005).

Asexual reproduction occurs quickly (seven hours) and done by binary fission of the filaments that break.

Multiple filaments of small dimensions are formed (Charpy et al., 2008; Jourdan, 2006). In this

phenomena of fragmentation there is a destruction of an intercalary cell, a sacrificial cell called necridium

(Figure 2). Necridia allow the formation of shorter segments or of hormogonia (Vonshak, 2002).

Figure 2: Linear filament in reproduction (magnification of 60 x).

4

This microorganism does not have a cell wall but rather a thin membrane, relatively fragile, made of four

layers of complex sugars (polysaccharides), with a major structural layer of peptidoglycan.

The central nucleoplasmic region appears to contain a number of ribosomes, cylindrical bodies,

carboxysomes and lipid droplets. The peripheral region of the cell also contains gas vacuoles and

several other subcellular inclusions like polyglucan and polyphosphate granules (Charpy et al., 2008;

Richmond, 2004).

1.1.4 Biochemical composition

Spirulina is very rich from a biochemical and nutritional point of view, having a significant amount of:

-Amino acids: the most significant, in terms of number, essential amino acids present in Spirulina are

isoleucine, leucine and valine and the most significant non-essential amino acids are glutamic and

aspartic acids (Henrikson, 1989; Moorehead, Capelli, & Cysewski, 2011b).

Isoleucine is needed for growth, intelligence development and nitrogen balance.

Leucine helps to increase muscular energy levels and stimulate brain function.

Valine assists with the co-ordination of muscular system as well as contributing to improved

mental capacity.

Aspartic acid helps with the transformation of carbohydrates to energy.

Glutamic acid, along with glucose, fuels the brain cells. It can also reduce the craving for alcohol

and stabilise mental health (Moorehead et al., 2011b; Tietze, 2004).

-Proteins: proteins correspond to about 60-70% of the dry weight of Spirulina. These proteins are easily

digested and quickly assimilated satisfying hunger very quickly because of the thin membrane. Thus,

the digestibility and adsorption are higher, fact that is very important for undernourished people (Adams,

2005; Henrikson, 1989; Moorehead et al., 2011b; Tietze, 2004; Vonshak, Torzillo, & Tomaseli, 1994).

Spirulina also contains enzymes, more precisely, the enzyme superoxide dismutase- SOD. This enzyme

catalyses the dismutation of superoxide radicals to hydrogen peroxide, protecting cells from toxic and

reactive oxygen species. Also, it may be involved in age-related degeneration (Moorehead et al., 2011b).

-Vitamins: particularly rare is vitamin B12 and provitamin A (retinol). It is important to mention that B12

is indicated in cases of fatigue, moodiness, pernicious anaemia and nerve degeneration (Henrikson,

1989; Moorehead et al., 2011b; Tietze, 2004).

-Minerals: such as iron that is used for making haemoglobin (the oxygen carrier in the blood) and

potassium that is used for regulating electrolytes. A deficiency in potassium can lead to heart attack and

muscular collapse.

5

- Lipids: Animal protein foods are high in calories, fat and cholesterol, however Spirulina as a source of

proteins is only five percent fat. The major lipids present in Arthrospira platensis are

monogalactosyldiacylglycerol (MGDG); sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol

(PG). Apart from that, this cyanobacterium has fatty acids such as omega-3 and γ-linolenic acid (GLA).

The latter is a rare polyunsaturated fatty acid (PUFA) that has been used for alleviating the symptoms

of premenstrual syndrome and for the treatment of atopic eczema. It is also a skin protector against UV

radiation, dehydration and activates the blood circulation in skin (Adams, 2005; Charpy et al., 2008;

Moorehead et al., 2011b; Tietze, 2004; Vonshak, 2002).

-Polysaccharides: formed by six neutral sugars such as fructose, rhamnose, mannose, glucose,

galactose and xylose. These microalgae have shown the ability to excrete polysaccharides to the growth

medium. Spirulina is also constituted by sulphate polysaccharides like calcium-spirulan (Ca-Sp) and

sodium spirulan (Na-Sp). The Ca-Sp has the capacity to inhibit replication of several virus and because

of this it can be a good candidate to fight HIV. It is also used in reducing cholesterol levels (Belay, 2002;

Moorehead et al., 2011b).

-Pigments: this cyanobacterium is very rich in pigments too (Figure 3). In its chloroplasts there are

pigments like chlorophylls, phycobilines like phycocyanin (with blue fluorescence) and phycoerythrin

(with red fluorescence), carotenoids (β-carotene and others) and xanthophylls (zeaxanthin,

myxoxanthophyll, cryptoxanthin, echinenone, fucoxanthin, violaxanthin and astaxanthin) (Charpy et al.,

2008; Henrikson, 1989; Moorehead, Capelli, & Cysewski, 2011a; Vonshak, 2002).

Chlorophyll a (Figure 4): this pigment is known as “green blood” because it is regarded as the

haemoglobin molecule in the human body. It is a phytonutrient responsible for cleaning and detoxifying

and is very beneficial for a healthy skin (avoids inflammations) (Domínguez, 2013; Henrikson, 1989;

Tietze, 2004; Vonshak et al., 1994; Vonshak, 2002).

Phycocyanin (C-PC) (Figure 5): it is the major component of the phycobiliprotein family. Phycocyanin is

a powerful water-soluble antioxidant blue pigment that gives Arthrospira platensis its bluish tint. It can

only be found in blue-green algae. C-PC is thought to help protect against renal failure and against

degenerative diseases like Parkinson and Alzheimer in rats. Recently, phycocyanin has showed itself

promising in treating cancer in animals, stimulate immune system and inhibit allergic inflammatory

response (Belay, 2002).

It is also used as a nutritive ingredient and natural dye in foods (dairy products, ice sherbets, jellies and

chewing gums) and cosmetics (Antelo, Anschau, Costa, & Kalil, 2010; Boussiba, 1979; Charpy et al.,

2008; Domínguez, 2013; Silveira, Burkert, Costa, Burkert, & Kalil, 2006; Various Autors, 2003).

Phycoerythrin (C-PE): is a large, red protein pigment complex accessory to the main chlorophyll

pigments. This pigment is very useful in laboratories for labelling antibodies in techniques of

immunofluorescence: fluorescent dyes for FACS analysis, for example.

6

Carotenoids: Carotenoids are used and stored in several parts of the body including the reproductive

system, the skin (gives it elasticity) and the retina.

β-carotene: Spirulina has been described as the richest food in β-carotene, an important antioxidant,

having ten times more β-carotene than carrots. This pigment has therapeutic effects like reducing cancer

risks and reducing cholesterol (Henrikson, 1989; Moorehead et al., 2011b; Tietze, 2004).

Zeaxanthin (Figure 4): is a very important antioxidant because it can cross the blood-brain barrier and

protect the eyes, brain and central nervous system and it does not become a pro-oxidant (Moorehead

et al., 2011b).

Figure 3: Extraction of all pigments present in Arthrospira platensis.

Figure 4: Extraction and separation between chlorophylls (green phase) and zeaxanthin (orange phase).

Figure 5: Extraction of phycocyanin present in Arthrospira platensis.

1.1.5 Applications

This microalga has been widely used as a source of food or dietary supplement for centuries. There are

reports that it was used as food in Mexico during the Aztec civilization at least some 500 years ago,

when Europeans arrived. Nowadays, it is still being used traditionally as food by the Kanembu tribe in

the Lake Chad area of the Republic of Chad in north-central Africa, where it is sold as dried bread called

“dihe” (Figure 6) (Belay, 2002; Moorehead et al., 2011b; Richmond, 2004; Vonshak & Richmond, 1988).

Apart from that, Spirulina has a high pharmaceutical interest as a source of active ingredients in several

areas. It can also be applied as adsorbent material for heavy metals.

Its production is relatively cheap and simple, because of its quick reproduction just requiring light and

inorganic nutrients, of its resistance to adverse environmental conditions and is easiness of harvesting.

Accordingly, Spirulina has a high interest in pharmaceutical, food and cosmetic industries, being already

world marketed in the form of powder, extrudate, pills and capsules.

7

In the late 1980’s and early 90’s, NASA included Spirulina as a food to be taken into space because it

grows fast, takes up very little space and its nutrient requirements can be met by recycled waste.

There are many programs to combat malnourished populations that use Spirulina, especially in Third

World villages (Heierli & Weid, 2007; Moorehead et al., 2011).

Figure 6: Women in Lake Chad working in sand filter to obtained “dihe”.

(http://huertosorganicos.com.mx/es-ES/servicios/cultivo-de-espirulina/item/17-nam-quam)

8

1.2 Cultivation Systems

There are many possible biotechnologies/systems used to cultivate and grow microalgae and

cyanobacteria at pilot scale. Some of them are open systems and others are closed systems.

Photobioreactors (closed systems) are best suited to the production of high value compounds: the

formation rate of the desired product can be enhanced by setting the proper culture conditions. Below

are described four of these systems.

1.2.1 Flat-plate photobioreactor

A flat-plate photobioreactor is a photobioreactor that consists in a thin rectangular box that can be made

of glass or transparent plastic, usually set vertically, so construction costs are relatively low.

These photobioreactors have important advantages for mass production of photoautotrophic

microorganisms and may become a standard reactor type best suitable for production of several

microalgae species since they can be oriented at different angles so as to modify the intensity of

impinging light and improve productivity. On the other hand, flat plate reactors, when compared with

tubular systems, can have a similar surface to volume ratio (high surface to volume ratios) depending

of the geometry of both systems. Considering flat plate reactors with aeration, then these

photobioreactors can also avoid O2 accumulation that can damage the cells.

In the flat plate PBRs used in this project, a temperature regulating coil and a diffuser (turbulent

streaming) were introduced. Generally the introduction of carbon dioxide occurs at the bottom of the box

to ensure that there is enough time to the interaction between gas and microalgae. With the diffuser it

is possible to control wall growth and biofouling. However, high energy is expended for mixing and

cooling. It is also introduced one syringe for collecting samples. Flat-plate photobioreactors are easily

scaled-up due to their modular design.

Flat plate photobioreactors (Figure 7) can be tilted towards the sun. Inclination angle allows an

optimization of the reception of the incident radiation ensuring higher areal productivity. (Qiang &

Richmond, 1996; Sierra et al., 2008).

Figure 7: Outdoor flat-plate photobioreactors.

(http://energy.gov/eere/bioenergy/production)

9

1.2.2 Raceway pond (RW)

Raceways are a type of system that consist in a recirculation channel in closed circuit, with typical depth

of 30 centimetres. These cultivation systems have depths that facilitate the distribution of solar light

since this is limited by optical absorption and by shelf-shading of microalgae. The mixture and circulation

are secured by an agitation system formed by a paddlewheel that operates continuously to avoid

sedimentation and biofilms formation; and the flow is orientated in turn of the baffles across the

channels, like is showed in Figure 8.

Figure 8: Representative scheme of a raceway pond to cultivate microalgae and cyanobacteria.

(https://wiki.uiowa.edu/display/greenergy/Algae+Biofuels#AlgaeBiofuels-RacewayPonds)

There are many materials that allow to construct raceways. Some raceways are made of packed earth

and others are made of concrete that can be coated with plastic.

The requirements of CO2 are usually met by CO2 present in the air, however, sometimes, aerators can

be installed to increase its absorption and avoid low CO2 transfer rates and consequent decrease of the

productivity of biomass.

Because raceways are a type of outdoor growing system, they cannot have effective control over

growing conditions, which can affect its productivity. The most critical factors are: evaporative losses,

temperature fluctuations over the course of the day and night, limitation of light and contamination by

other algae and microorganisms (fungi, bacteria, protozoa and others). However, raceway ponds were

in 2003 used by 98% of commercial microalgae production systems (Benemann, 2013). The limitation

of light due to the thickness of the top layer also affects productivity.

These cultivation systems are low cost systems for a large scale production that not necessarily compete

with the agricultural land as a result of its easier less implementation and operation and its longer

durability. Raceway ponds design often depend on the local conditions (Richmond, 2004).

In addition, they have a low energy requirement and its maintenance and cleaning are easy.

10

1.2.3 Cascade raceway (CRW)

The cascade raceways have a design that was previously developed by A4F for operation in desert or

rainy climates.

In cascade raceway systems, turbulence is created by gravity. The culture suspension flows from the

top to the bottom of sloping surfaces. This type of system allows adoption of very thin culture layers.

Similarly to RW, these systems are limited by several problems such as sedimentation of the cells at

certain points with low turbulence, strong evaporative losses or even photo inhibition if the culture is

less concentrated and the width of ramps is high (Figure 9).

Figure 9: Cascade raceway set-up.

(http://www.botany.ut.ee/kaitsmised_2011/Karin_Ojamae_mag.pdf)

In this cultivation system there is usually a tank where the culture is stored, for example, when rainfall

is very high. This tank is associated with a recirculation pump that aims to pump the culture again to the

higher ramp, giving rise to a new cycle of the culture circuit on the ramps. It is very important to have

control on the type and the work frequency of the pump to avoid shear stress of fragile microalgae, such

as Spirulina.

11

1.2.4 Tubular photobioreactor (PBR)

This type of cultivation system is a closed system consisting of tubes made of transparent materials,

holding and allowing the microalgae to use light. The tubular array can be aligned horizontally or

vertically. Tubes generally have diameters up to 10 centimetres. The diameter of the tubes is limited

because light cannot penetrate in depth in the culture due to its concentration (Figure 10).

The ground below the tubes is often painted with white colour or covered with white plastic to increase

albedo. A high albedo increases the total light received by tubes.

Typically, in these systems, culture from the tank is conducted to the tubes using a pump which should

work on a frequency that does not cause shear stress and therefore damage the microalgae cell walls

of more fragile species. For cultivations of microalgae with a silicate wall, the pump can work at higher

frequencies due to the resistance imposed by the silicates of these walls. In this type of reactor

turbulence is driven by the pump (mixing technique), to avoid the formation of biofilms.

The culture must return periodically to the tank, where it is allowed to eliminate the accumulated oxygen

that can be harmful.

Along the tubes, the pH of the culture biomass increases due to the consumption of CO2. CO2 is added

in the central tank in response to a pH controller. Additional injection points can be necessary to avoid

the limitation of carbon and the excessive increase of the pH.

Figure 10: PBR with horizontally displayed tubes.

(http://www.a4f.pt/gallery.html)

Table 2 below summarizes the advantages and the limitations of the open and closed cultivation

systems.

12

Table.2: Comparison between open ponds and closed photobioreactors

Parameter Open Closed

Control of process parameters Low High

Contamination risk High Low

Water loss due to evaporation High Low

CO2 loss High Low

O2 build-up Low High

Weather dependence High Low

Cost per area Low High

Energy required Low High

In conclusion, choosing a photobioreactor depends on the cultivated species, the location for the culture

system and the desired final product.

1.3 Areal Productivity vs Volumetric Productivity

It is essential to define the concept of productivity and what is the difference between areal and

volumetric productivities.

Several options exist for considering the area associated to areal productivity (AP). It can be the biomass

productivity per unit of ground area occupied by the reactor. In this way the amount of biomass is

quantified in dry weight (g DW L-1) and AP is expressed as g.m-2.day-1. On another concept, the

productivity per unit of ground area of the reactor that is directly exposed to solar radiation. In this thesis,

the second case was considered.

The volumetric productivity (VP) is the biomass productivity per unit reactor volume expressed as g.L-

1.day-1. The VP is a very important key to understand how efficiently the unit volume of the reactor is

used.

Both productivities can be compared together to draw conclusions, because for the same area (same

areal productivity), the volumes of different cultivations systems can be different (different volumetric

productivity).

13

According to the objective, the different cultivation systems should not be viewed as competing, but

complementing technologies since they present different volumetric and areal productivities between

them. This fact could be useful in microalgae industry (Richmond, 2004; Wolf et al., 2016).

1.4 Culture Medium vs Nutritive Medium

The culture medium is the support medium to maintain the viability of the microorganisms present in a

certain biological sample. A culture of microalgae contains the culture medium and the microalgae cells.

The culture medium is usually an aqueous solution of mineral salts.

In this thesis, nutritive medium refers to a concentrated solution that contains the necessary nutrients

for reproduction/multiplication of microalgae of a culture. The principal compounds present in the

nutritive medium are nitrogen, phosphorous and other mineral salts.

1.5 Harvesting of Biomass

All downstream processing of cultures of microalgae/cyanobacteria involves one or more steps to

promote solid-liquid separation, because biomass needs to be separated from the culture medium. Also,

it can be used to separate the liquid phase from the cell debris following cell disruption for release of the

metabolites of interest.

Biomass is usually harvested by mechanical, chemical and electrical-based methods, for example:

centrifugation, sedimentation, filtration and sometimes an additional step of flocculation is required

(chemically or electrically induced) (Barros, Gonçalves, Simões, & Pires, 2015; Ganesan V., 2014;

Grima, Fernández, & Medina, 2005; Richmond, 2004).

Biological based methods such as the use of planktivorous fish (e.g. tilapia) are also currently being

investigated as a means to reduce harvesting cost that has been reported to account for 15-30% of the

production costs. In this process, the microalgae are then batch fed to caged fish and the fish droppings

and any sedimented microalgae are brought to the surface on an inclined conveyer belt to be fed to an

anaerobic digester (Christenson & Sims, 2011).

14

1.5.1 Centrifugation

Almost all types of microalgae can be separated from the culture medium by centrifugation.

Centrifugation is the most reliable and the fastest harvesting method. The equipment used to do this

operation is the centrifuge that is basically a sedimentation tank with enhanced gravitational force to

increase the rate of sedimentation. On the other hand, the exposure of microalgae cells to high

gravitational and shear forces result in cell structural damage and therefore centrifugation should not be

used for recovering biomass in which cell integrity must be maintained throughout the harvesting

process (Figure 11).

This equipment can be sterilized and easily cleaned, however it is the most expensive harvesting

technique because it requires a great amount of energy per volume of culture for high cell removal

efficiency. To reduce the energy consumed in the process, the efficiency of harvesting of biomass can

be sacrificed for greater process volumes with lower energy consumption (Barros et al., 2015; Pires,

2015).

Figure 11: Microalgae biomass harvested by centrifugation from a culture.

(http://www.evodos.eu/high-quality-output-2/)

1.5.2 Sedimentation

Sedimentation is a process with low costs that allows obtaining concentration of solids of about 1.5%. It

is relatively slow process since it depends of the sedimentation time which may cause biomass to

deteriorate during settling.

On the other hand, sedimentation time depends of several factors such as microalgae size and density

and viscosity (culture temperature). Normally, sedimentation is preceded by a flocculation step to

increase final concentration values and make the process faster (Richmond, 2004).

15

1.5.3 Filtration

Filtration is a common method of solid-liquid separation that it is used as a dewatering process in case

of microalgae harvesting.

Microalgae require the use of membrane microfiltration, which has a nominal pore size ranging from 0.1

to 10 µm. On the other hand, to recover microalgae relatively large or microalgae with tendency to form

aggregates, macrofiltration is more indicated, while to recover metabolites one should use ultrafiltration.

In all these filtration techniques, to force fluid flow through a membrane, the maintenance of a pressure

drop across the system is required. There are two filtration methods: dead-end and tangential flow

filtration.

A decrease in filtration flux upon a constant pressure difference and an increase of resistance is

observed because of the increase of microalgae deposits over the membrane, clogging the membrane.

This effect is most visible in membranes of ultra and microfiltration that tend to clog more easily. Because

of that fact, these membranes require a regular (automated) cleaning.

Filtration allows the complete separation of cells and other contaminants present in culture from the

culture medium which can be a great advantage for harvesting biomass in a system with recirculation

of culture medium (Ganesan V., 2014; Grima et al., 2005; Richmond, 2004).

1.5.4 Flocculation

Flocculation is the process to promote the collection of cells into aggregates that offer many advantages

by facilitating cell/broth separation. There are different ways to induce flocculation: by adding a

biopolymer- bioflocculation; changing the pH that frequently causes the spontaneous flocculation of

microalgae cultures- autoflocculation; or adding an electrolyte (chemical flocculants).

Chemicals reduce the cell surface charge and form precipitates that enhance the clustering and

sedimentation processes. Depending on the downstream process, the appropriate chemical species is

selected. This process has the disadvantage of requiring medium treatment for flocculants removal

before its reintroduction into the culture, creating extra operational costs (Pires, 2015; Richmond,

2004).The harvesting of microalgae is likely to remain an active area of research.

Nowadays, a universal harvesting method does not exist, however experience has demonstrated that

for all species it is possible to develop an economical, appropriate and adapted method to the

requirements of microalgae harvesting system: salt concentration, strain features, cell damage and

contamination.

Furthermore, the selected harvesting method should also allow recycling of culture medium.

16

1.6 The Strategy of Recirculation of Culture Medium

Culture medium recirculation is by definition the process by which culture medium, after harvesting of

the biomass, is reintroduced in the cultivation system. In this way, the medium recycling strategy is a

simultaneous process of culture dilution and recycling of culture medium. This process allows the

utilization of previously unconsumed nutrients and the saving of a great amount of water.

When this strategy is not applied, all the removed culture medium is rejected which implies that the

same amount of fresh medium must be introduced in the system. Failure to use this strategy leads to

increased cultivation costs due to the large amount of reagents, nutrients and water used.

In large scale microalgae cultivation, the reuse of culture medium becomes essential, particularly in

culture media which involve and require special conditions/compounds and, consequently, higher

expenses (Gaspar, 2014).

On the other hand, this strategy frequently leads to a loss of productivity, which is thought to be related

to nutrients and metabolites ratios in the medium which might be altered by reactions occurring during

cultivation, leading to toxic metabolite build-up that can be a limiting growth factor when present in

excessive concentrations. Other factors that can be related to the productivity loss are: cellular debris

accumulation (e.g. plasmatic membrane released to culture medium after cellular lysis may induce the

aggregate formation and trapping microalgae cells inside) or predator contaminations (e.g. protozoa,

fungi and bacteria).

Considering all these aspects, recirculation strategy must be carefully tested and optimised for each

specific microalgae species and system conditions and the harvesting strategy to separate cells from

culture medium should be optimised in order to avoid the accumulation of organic matter in the culture

(Depraetere et al., 2015; Rodolfi, Zittelli, Barsanti, Rosati, & Tredici, 2003; Yang et al., 2011).

Figures 12 and 13 compare the flow diagrams of a microalgae cultivation process without and with

recirculation of the culture medium, respectively, where arrows represent the materials flux and the

boxes represent industrial processes (Gaspar, 2014).

Figure 12: Scheme of microalgae production process operating without recirculation of culture medium.

17

Figure 13: Scheme of microalgae production process operating with recirculation of culture medium.

1.7 Drying of Biomass

After harvesting of microalgae biomass, which should result in a 50-200 fold concentration, the biomass

(5-15% dry weight) must be quickly processed. The principal objective of the drying process is to avoid

spoilage of the final product and to extend its shelf life. The most common methods to dry microalgae

are: spray-drying, freeze-drying/lyophilisation, drum-drying and sun-drying.

1.7.1 Spray-drying

Spray-drying is a drying method that should be preferably used to process higher value products in

which microalgae cells must be kept intact (Figure 14).

This method allows a rapid and continuous drying of emulsions, solutions and slurries which involve

spraying atomised solution droplets into a vertical large tower where they are continuously in contact

with hot air.

Small droplet size and large surface area guarantee high evaporation rate that allows a complete drying

within few seconds.

Although this drying method may be considered appropriate for production of microalgae and

cyanobacteria for human food (ex: Spirulina platensis), spray-drying can cause deterioration of biomass

components, such as proteins or pigments (Grima et al., 2005; Richmond, 2004).

18

Figure 14: Aspect of Spirulina dried in spray-dyer.

1.7.2 Freeze-drying

Lyophilisation is another method for drying biomass. In this process, microalgae slurries are frozen and

the ice crystals are sublimed afterwards. This phenomena of sublimation of the ice crystals allows the

formation of numerous cavities through which water can penetrate, enabling the possibility of quickly re-

hydrating lyophilised biomass.

Freeze-drying involves high operation costs and expensive equipment, so it is only recommended to

dehydrate biomass when maintaining the functionality of the biomass components or biochemical

activity is fundamental.

1.7.3 Drum-drying

Drum-drying is a method used for drying out liquids from raw materials, in this case, microalgae biomass.

The biomass is dried at relatively low temperatures over rotating drums and the product is milled to a

finished flake or powder form.

This technique has both the advantages of drying viscous raw materials that cannot be easily dried with

other methods and be easily cleaned and operated.

Drum-drying is considered the most adequate drying method for preparing animal-grade biomass

(Grima et al., 2005).

19

1.7.4 Sun-drying

Sun-drying is a technique that uses solar energy to dry substances, especially food (Figure 15). The

sun-dryer has a black absorbing surface which collects the light and converts it to heat. To increase

efficiency, these driers may have enclosures, glass covers and vents.

The major difficulty to use sun-dryer is the high water content present in microalgae biomass, however

the sun-dryers are the most inexpensive alternative for drying biomass (Grima et al., 2005).

Figure 15: Aspect of Spirulina dried in sun-dryer.

20

21

Chapter 2:

Framework and Goals

Nowadays, the creation of a productive and sustainable cultivation system to grow microalgae is one of

the biggest challenges of microalgae biotechnology. Arthrospira platensis with its large potential, as

mentioned in chapter 1 of this dissertation has become a great target of study and research.

Thus, the present work describes the production of Spirulina using different production technologies:

flat-plate photobioreactor; cascade raceway and conventional raceway at pilot scale using as initial

cultivation medium SAG- a standard culture medium with many nutrients and high pH- but with some

steps of optimization. Therefore, productivity parameters for the three cultivation systems were analysed

throughout time of cultivation.

On the other hand, it is very important to find strategies to reduce nutrient supply cost and water usage,

since all cultivation systems mentioned use a considerable amount of nutrients and water. Hence, the

main goal of this work was analysing the influence of the recycling of the culture medium on a pilot scale

Arthrospira platensis cultivation for producing biomass. Medium recycling is fundamental to ensure cost-

effectiveness and sustainability. Furthermore, medium recycling is also a source of productivity loss due

to nutrient imbalance and organic matter and contaminants/inhibitors accumulation.

To do so, a comparison between the chemical composition of fresh culture medium and the chemical

composition of the culture medium after three medium recirculation cycles was performed. The final goal

was to adjust the recipe of culture medium after several rounds of trials.

A comparison between culturing Arthrospira platensis using fresh culture medium and formulated

seawater as culture medium was made with the objective of studying if formulated seawater can be a

good and economical alternative.

It must be stressed that a characterization of biochemical profile of the cyanobacteria under different

production conditions was conducted to observe the behaviour of Spirulina and understand which are

the better conditions to obtain a certain product of interest, for example, protein.

The project was developed in the laboratory and pilot scale unit of A4F – Algae for future in Lisbon.

22

23

Chapter 3:

Materials and Methods

3.1 Reagents and Solutions

Table 3: List of reagents used in this work and their respective supplier.

Use Reagent Supplier

Culture medium recipe

Na2CO3 BicarFCC Solvay

K2SO4 CHEM-LAB

NaHCO3 Soda Solvay Light

Nutritive medium recipe (A)

NaNO3 Laborspirit

Na2H2C10H12O8·2H2O VWR

KH2PO4 JMGS

MgSO4·7H2O Acofarma

ZnSO4·7H2O José M. Vaz Pereira SA

MnCl2 Scharlau

Na2MoO4·2H2O PROLABO

CoCl2·6H2O Panreac

CuSO4·5H2O JMGS

FeSO4·7H2O CHEM-LAB

Protein quantification

method

KNaC4H4O6·4H2O Scharlau

CuSO4 JMGS

Na2CO3 CHEM-LAB

C6H6O (Folin & Ciocalteu’s

phenol)

BioCHEM

24

Table 4: List of reagents used in this work and their respective supplier.

Pigments quantification

method

NaOH (2M) EKA

C3H6O (99%) Panreac

n-C6H14 (98,5%) Carlo Erba

CH3OH (99%) Carlo Erba

H3PO4 (Phosphate buffer

(0,1M)) Fisher Scientific

3.2 Equipment

Table 5: Equipment used in the project and its respective model and manufacturer.

Equipment Model- Manufacturer

Autoclave Uniclave 88, AJC

Precision balance (± 0,0005 g) Ohaus

Dry weight balance (± 0,0005 g) AND MS-70

Vacuum Pump Comecta

Centrifuge Hermle Z 400 K

Spectrophotometer UV – Vis (± 0,005

AU)

Genesys 10S UV-Vis- Thermo Scientific,

US

Portable conductivity and pH-meter Mettler Toledo

Optical Microscope Microscope Olympus BX53

Portable Refractometer ZUZI

Tubes for biomass determination VoluPAC, Sartorius Stedim

Vortex Vortex GENIE 2, Scientific Industries

3.3 Biological Material

The cyanobacterium selected for this work was Arthrospira platensis. The strain was kept isolated and

free of contaminants in the algae collection of A4F.

25

3.4 Cultivation Systems

The cultivation systems used in this research were flat-plate photobioreactors (inside and outside of

greenhouse) and raceways- conventional and cascade. All these systems were described in chapter 1.

The first experiment took place in a flat-plate photobioreactor inside of a greenhouse between March

16th and April 26th. The reactor was inoculated with 20L of lab culture from 4 flasks of the A4F-Lisbon

Laboratory maintained in stock. To make up the work volume of 60L, 40 L of culture medium were added

(Figure 16).

In this system, the maximum temperature set-point was defined as 25ºC and was controlled by a coil

thermoregulation system which cools the reactor wall very quickly through its constant water flux.

The uniform mixing was established through a diffuser in the bottom of the reactor which was the air

supply of the system. The bottom location of the diffuser provides a uniform mixing of all parts of the

reactor and prevents accumulation and deposit of biomass.

The injection of pure CO2 pulses, allowed the real time pH control between 9 and 10.

To avoid the photoinhibition phenomenon, i.e. to avoid the excess of photons incidence on cells when

cultures are very diluted, a shading net was used to cover part of the reactor.

Figure 16: Flat-plate photobioreactor used and located inside of greenhouse (day 0- after inoculation).

26

The second experiment took place in a cascade raceway between March 23th and March 29th. This

reactor was located outside of a greenhouse and was inoculated with 50L of culture from flat-plate

photobioreactor of the first experiment when the first renewal was done. Apart from that, 550L of culture

medium were added to obtain a work volume of 600L (Figure 17).

The culture pH set-point was maintained between 9 and 10 and the temperature was controlled by

evaporation.

Every day, the volume evaporated was replaced using tap water, maintaining the work volume constant.

In this system, as mentioned in section 1.2.3, there is a recirculation pump that aims to pump the culture

again to higher ramps. The culture height on the ramps was 3 centimetres.

Figure 17: Cascade raceway used (day 0- after inoculation).

The third experiment took place in a flat-plate photobioreactor located outdoor between April 15th and

May 19th. This reactor is identical to the one described in the first experiment and the conditions

(temperature and pH set-points) were maintained (Figure 18).

Figure 18: Flat-plate photobioreactor used and located outdoor (day 0- after inoculation).

27

The fourth experiment took place in the same cascade raceway used in the second experiment. This

assay occurred between April 26th and May 4th.

In this assay, some adjustments were done. The reactor was inoculated with total volume of flat-plate

photobioreactor used in first experiment (60L). The work volume was fixed in 300L. To avoid the

photoinhibition phenomenon, the width of ramps was decreased to allow an increase of the culture

height on the ramps to 7 centimetres (Figure 19). Every day the volume evaporated was replaced by

tap water.

The temperature was controlled by evaporation and pH set-point of the culture was maintained between

9 and 10.

While in the first assay that occurred in this cultivation system, the recirculation pump worked with an

increment of work frequency until 21 Hz, in the second assay the recirculation pump frequency was

maintained constant at 19 Hz.

On May 4th all volume of the cascade raceway was harvested by filtration and the concentrated biomass

was dried in a spray-dryer.

Figure 19: Cascade raceway used after some adjustments (day 0- after inoculation).

28

The last experiment took place in a conventional raceway between May 19thand June 24th. The reactor

was inoculated with all the volume from flat-plate photobioreactor of the third experiment (60L). To obtain

a work volume of 670 L, 610 L of culture medium were added. This work volume corresponds to a culture

height on channels of 11.5 centimetres (Figure 20).

The temperature was controlled by evaporation and the culture pH set-point was maintained between 9

and 10. Every day the volume evaporated was replaced by tap water.

The mixture and circulation are secured by a recirculation paddlewheel that operates continuously.

Figure 20: Conventional raceway used (day 0- after inoculation).

Table 5 below summarized all the cultivation conditions of the different cultivation systems studied.

29

Table 6: Summary of cultivation conditions of the all cultivation systems.

Cultivation systems Indoor Flat-plate

photobioreactor

Cascade raceway

(1st Assay)

Outdoor Flat-plate

photobioreactor

Cascade raceway

(2nd Assay)

Conventional Raceway

Date of assay start March 16th March 23th April 15th April 26th May 19th

Date of assay end April 26th March 29th May 19th May 4th June 24th

Initial dry-weight (g/L) 0.188 0.100 0.319 0.139 0.440

Culture volume / Work

volume (L)

60 600 60 300 670

Maximum temperature set-

point (ºC)

25 Controlled by

evaporation

25 Controlled by

evaporation

Controlled by evaporation

pH set-point 9-10 9-10 9-10 9-10 9-10

Nitrogen set-point (mM) 6 6 6 6 6

Culture height (cm) - 3 - 7 11.5

Renewals (%) (v/v) First: 83% (March 22th)

Second: 80% (April 15th)

- First: 30% (May 5th) - First: 54% (May 31st )

Second: 54% (June 7th)

Third: 50% ( June 16th)

Recirculation/ Harvesting of

culture medium and

biomass

Harvesting of all the

culture in the end of

the assay

Three recirculation of culture

medium of 80% (v/v)

30

3.5 Culture Medium Formulation

The culture medium used to cultivate Arthrospira platensis in A4F cultivation systems is the result of an

optimisation work done in the company. The initial culture medium used for this optimization (SAG

medium) results of a mixture between two different solutions: SPIR-1 and SPIR-2. In Table 6 it is

possible to observe the components and concentrations of each of them.

Table 7: Composition of culture medium reference recipe: SAG medium. Modified from (Aiba & Ogawa, 1977)

Reagent Concentration (mM)

NaHCO3 162

Culture

Medium

Na2CO3 38

K2HPO4 2.9

NaNO3 29.4

Nutritive

Medium

K2SO4 5.74

NaCl 17.1

MgSO4·7H2O 0.81

CaCl2·2H2O 0.27

P-IV Metal

solution

6 mL/ 0.5 L

Na2EDTA·2H2O 2

FeCl3·6H2O 0.36

MnCl2·4H2O 0.21

ZnCl2 0.037

CoCl2·6H2O 0.0084

Na2MoO4·2H2O 0.017

31

Table 8: Composition of culture medium reference recipe: SAG medium. Modified from (Aiba & Ogawa, 1977)

Chu

Micronutrient

solution

1 mL/ 0.5 L

Nutritive

Medium

CuSO2·5H2O 0.08 µM

ZnSO2·7H2O 0.15 µM

CoCl2·6H2O 0.084 µM

MnCl2·4H2O 0.061 µM

Na2MoO4·2H2O 0.052 µM

H3BO3 10 µM

Na2EDTA·2H2O 0.13 µM

Vitamin B12

1 mL/ 0.5 L

HEPES buffer pH 7,8 2.4 g/ 200 mL

distillate H2O

Vitamin B12 0.027 g/ 200 mL

distillate H2O

In A4F’s laboratory, as mentioned, an initial assay was performed to optimize this culture medium,

analysing which compounds are not essential for Spirulina growth and that contribute to the costs

associated. With this objective in mind, the company concluded that the three components that make

possible Arthrospira platensis’s growth are as follow: sodium bicarbonate (NaHCO3), sodium carbonate

(Na2CO3) and potassium sulphate (K2SO4).

Each of these reagents have an important role to contribute to an ideal growth of Spirulina:

NaHCO3 and Na2CO3: are reagents that contribute to maintain the pH between 9 and 10, ideal

range for Spirulina, due to their pH buffer. On the other hand, these reagents are a carbon

source for Spirulina growth.

K2SO4: it is the source of potassium and sulphur necessary to grow Spirulina which do not exist

in sufficient quantities in regular water sources (Jourdan, 2006). Previous assays done in A4F

showed that Spirulina cannot grow without potassium sulphate.

In order to protect the intellectual property of A4F, the reagents concentration in the optimized recipe

of Spirulina culture medium are not presented. Instead, Table 7 provides ranges of variation in the

concentration of each reagent between the reference recipe and the recipe developed by A4F for

culture medium.

32

Table 9: Comparison between the reference and recipe developed by A4F according to the optimization assay performed.

Reagent Δ (%)

NaHCO3 [+ 15]

Culture

medium

Na2CO3 [+ 15]

K2SO4 [+ 15]

NaNO3 [+ 25]

Nutritive

medium

KH2PO4 [+ 25]

Disodium EDTA [+ 25]

MgSO4·7H2O [+ 25]

FeSO4·7H2O [+ 25]

ZnSO4·7H2O [+ 25]

MnCl2·4H2O [+ 25]

Na2MoO4·2H2O [+ 25]

CoCl2·6H2O [+ 25]

CuSO2·5H2O [+ 25]

This culture medium was accompanied with the nutritive medium (see Section 3.1, Table 3) with the

desired nitrate concentration. The concentration of nitrate never was limited since it was regularly

measure. It is important to mention that the nutritive medium does not contain vitamins.

33

3.6 Operational Procedures

3.6.1 Inoculation

First, it is important to define the concept of inoculation. Inoculation is the act of transferring the culture

from the existing cultivation chamber in the laboratory to the culture systems located outdoors. This

procedure, from a scientific point of view, consists in the initial set-point of growth conditions where all

conditions must be identical in order to guarantee the reliability of the results.

The lab inoculum culture grew on culture medium described in Table 3.5 and was complemented with

the nutritive medium (see Table 3.1), and was maintained in linear growth phase through periodic

renewals.

The cultivation systems used were previously disinfected with sodium hypochlorite 13% (v/v).

All the reactors were inoculated with direct transfer of the laboratory culture and after inoculation, the

differentiated growth conditions started to be applied.

3.6.2 Renewal

The renewal process consists in replacing a certain fraction of the total culture system volume harvested,

with fresh culture medium and nutrients.

In the work developed, this procedure was conducted when the culture entered in stationary phase and

the renewal rate (rate of volume removed from the culture system) was determined case by case.

The renewal process can be done in different ways: (1) without recirculation of the exhaust culture

medium after harvesting of biomass- in this case, as mentioned, the volume replacement is done by

fresh culture medium; or (2) with direct recirculation of the culture medium after harvesting of biomass-

there is exhaust culture medium recycling).

3.6.2.1 Renewal without recirculation

According to the desired rate of renewal, the culture volume was removed with the aeration system

turned off. The fresh culture medium volume was added to the remaining volume and the nutritive

medium was added in accordance to the intended nitrate ion concentration.

34

3.6.2.2 Renewal with direct recirculation

This operational procedure also followed the previously described first step of renewal without

recirculation (see Section 3.6.2.1).

After this step, the culture medium was filtrated through a microfiltration membrane system and the

exhaust culture medium volume was added back to the cultivation system.

Since the yield of microfiltration is not 100%, a certain volume of fresh culture medium was added for

make-up, as well as the nutritive medium in accordance to the intended nitrate ion concentration.

3.6.3 Collecting culture medium for elemental analysis

This procedure was applied when it was necessary to collect culture medium for external elemental

analysis.

The culture medium harvesting took place in two different phases: the fresh culture medium used for

inoculation; and permeate obtained after filtration in each recirculation culture medium.

The culture medium of each phase was carefully transferred to sample plastic flasks properly identified

and stored at -18ºC and sent for external analysis.

3.6.4 Collecting of biomass for biochemical analysis (protein analysis)

This operational procedure was applied to collect biomass and perform biochemical assays to compare

the drying processes using the spray-dryer and using the sun-dryer.

The biomass harvesting took place in different phases of the recirculation culture medium assay. It is

very significant to mention that the biomass was washed by diafiltration in the same membrane filtration

system used, to remove all salts present in the culture medium.

Afterwards the biomass was stored in a chest-freezer at -20ºC until analysed.

35

3.6.5 Drying of biomass in spray-dryer vs drying of biomass in sun-dryer

In section 3.6.4 it was mentioned that a sampling of biomass was performed to compare the drying using

the spray-dryer or sun-dryer. Below, in Table 8, the principal differences between the two methods to

dry Arthrospira platensis are summarized.

Since one of the methods used to drying biomass uses higher temperatures, it is essential to understand

if at these temperatures there is maintenance of intracellular compounds such as proteins and lipids.

There are several papers in the literature that can support that the total lipids content does not suffer

alterations using diverse drying methods. However, the drying methods can modify the colour and

morphological characteristics of the dried product (Pinheiro, Strieder, & Pinto, 2016).

36

Table 10: Comparison between spray-dryer and sun-dryer

Equipment

Temperature

of drying

(ºC)

Exposure/

Time of

drying

Observations Photos

Spray-dryer 60a 3 seconds

The

atomization is

instantaneous.

Biomass

obtained in

form of

powder.

Sun-dryer 40-50 14-17hb

Drying done

during night

period (5.pm -

10. am).

Biomass

obtained is

crunchy.

a (Vonshak, 2002); b (Prakash et al., 2007; Tiburcio, Galvez, Cruz, & Gavino, 2007)

To better compare the different drying methods it would be interesting to perform energy calculations

for each process

37

3.7 Analytical Methods

3.7.1 Microscopic observation

This is a quick method that allows monitoring the development of the cultures and the presence of

contaminants.

The cultures were observed under the microscope in two distinct phases of observation whenever the

sample collection was done. First, the culture was observed through the lower capacity objectives (10x

and 20x) in order to search for contaminants of great dimensions (such as rotifers), crystals of culture

medium and possible microscopic debris. Afterwards, the culture was observed with higher capacity

objectives (40x and 60x) to search for contaminations of lower proportions like bacteria and small ciliates

and to observe the general health state and some organelles of the cells.

3.7.2 Determination of culture concentration

One of the basic parameters for monitoring the performance of microalgae production systems is the

estimation of the biomass produced. The growth of microalgae and cyanobacterium cultures can be

expressed in several ways such as the increment of biomass, the number of cells, the amount of

pigments, proteins and other components over a given period of time.

In this research, biomass was estimated throughout optical density (OD) measurements using a

wavelength selected according to the cyanobacterium pigments; dry weight and packed cell volume.

Thereafter, the values obtained were correlated between each one of them.

3.7.2.1 Determination of optical density (OD)

Using a UV-Vis spectrophotometer, cell growth was monitored by measuring the OD at a wavelength of

730 nm. Each sample was read in duplicate in plastic cuvettes, with 1 cm of path, against fresh culture

medium, to obtain accurate results.

In a later phase of Arthrospira platensis cell cultures, a dilution (with fresh culture medium) of 1:15 of

the samples was required to respect the linearity of the Beer-Lambert law.

The OD values were determined three times per week during this study. Every time that OD was not

measured, a linear interpolation was performed in order to determine the missing values.

38

3.7.2.2 Dry weight (DW)

The growth and development of the culture in test was also assessed through the biomass dry weight,

where a direct correlation between the light absorption and dry weight at different concentrations was

established.

For this method the moisture analyser was used to heat the sample at 180 ºC and measure the dry

weight in g/L (Equation 1). The samples of the cultures were filtrated, using 1.2 µm diameter filters

(Microfibre Filter Paper) and washed with demineralized water to remove the salts of the culture medium,

in pre-weighed filters and then dried in the moisture analyser.

𝐷𝑊 (𝑔. 𝐿−1) =(𝑚𝐹 − 𝑚𝐼)

𝑉𝑜𝑙

Equation 1: Determination of dry weight

where,

DW: dry-weight in g/L

mF: mass of biomass and filters after filtration in g

mI: mass of filters before filtration in g

Vol: volume of the sample dried in L

3.7.2.3 Packed cell volume (PCV)

The PCV is a method that allows to quantify the amount of existing biomass in a given sample volume.

The volume of sample was inserted in a specific centrifuge tube that was centrifuged at 4500 rpm during

60 seconds (Minispin Centrifuge). The biomass was compacted by the centrifugal force, obtaining a

volume of biomass pellet (Figure 21).

A percentage between the volume of biomass obtained (that can include contaminants and other

material) and the volume of sample inserted gave a notion of the existing biomass in the cultures.

Figure 21: Biomass obtained by PCV.

39

3.7.2.4 Correlations for determining culture concentration

The correlations between DW (g L-1) and OD730, between PCV (%) and OD730 and between DW (g L-1)

and PCV (%) of Arthrospira platensis cultures are presented in Figures 22, 23 and 24 respectively.

All the calibration curves were obtained by linear regressions from measurements of the culture samples

of different culture systems (see sections 3.7.2.1 at 3.7.2.3).

Figure 22: Calibration curve between OD730 measured by the spectrophotometer and the DW measured at 180ºC

in the moisture analyser for Arthrospira platensis.

.

Figure 23: Calibration curve between OD730 measured by the spectrophotometer and the PCV obtained by

centrifugation for Arthrospira platensis.

y = 0.6809xR² = 0.9763

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0.0 1.0 2.0 3.0 4.0

DW

(g/

L)

OD730nm

Correlation DW vs OD

y = 1.0402xR² = 0.9688

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

PC

V (

%)

OD730nm

Correlation PCV vs OD

40

Figure 24: Calibration curve between PCV obtained by centrifugation and the DW measured at 180ºC in the

moisture analyser for Arthrospira platensis.

All linear regressions above (see Figure 22, 23 and 24) have been obtained by plotting all the points

obtained for the different cultivation systems under study. Several series of the data are shown to

understand if different cultivation systems can be correlated be the same linear equation: each series

was represented by a different colour and for a cultivation system.

It was observed that the points obtained are well-adjusted by straight lines obtained, so they were

represented in a single series.

R-squared (it is also known as the coefficient of determination) is a statistical measure of how close the

data are to the fitted regression line, so in general, the higher the R-squared, the better the model fits

the data (model explains better the variability of the response data around its mean).

The worse R-squared, as it is possible to observe, occurs for correlations with PCV.

First of all the PCV is a method that compacts biomass present is a sample that includes, in addition to

Spirulina cells, contaminants like bacteria and other compounds. These facts can lead to a certain

uncertainly of some measurements done especially in samples of cultivation systems localized outside

the greenhouse, where the risk of contaminations is higher. However, this fact can also influence the

other techniques used since they also have into account the contaminants present in samples.

There is one reason that can explain these values of R2: A reading of the volume of biomass pellet

obtained in a graduated PCV tube is done by the operator (see Figure 21), there is associated an error

of measure. This error may also contribute to the R2 obtained.

y = 0.6134xR² = 0.9401

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0.00 1.00 2.00 3.00 4.00

DW

(g/

L)

PCV (%)

Correlation PCV vs DW

41

3.7.3. Determination of the culture volumetric productivity

Culture volumetric productivity was calculated for each cultivation day and according to Equation 2 was

determined in (g DW. L-1.day-1)

𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝑝𝑟𝑜𝑑𝑢𝑐𝑒𝑑 𝑏𝑖𝑜𝑚𝑎𝑠𝑠(𝑔)

𝑉𝑐𝑢𝑙𝑡𝑢𝑟𝑒 (𝐿) × 𝑡𝑐𝑢𝑙𝑡𝑖𝑣𝑎𝑡𝑖𝑜𝑛 (𝑑𝑎𝑦𝑠)

Equation 2: Determination of culture volumetric productivity.

where,

Vculture: volume of the culture present in the cultivation system in L

tcultivation: time passed in days to obtain a certain produced biomass

3.7.4 Determination of the culture areal productivity

Culture areal productivity was calculated for each cultivation day and according to Equation 3 was

determined in (g DW. m-2.day-1).

𝐶𝑢𝑙𝑡𝑢𝑟𝑒 𝑎𝑣𝑒𝑟𝑎𝑔𝑒 𝑝𝑟𝑜𝑑𝑢𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = 𝑉𝑃 (𝑔𝐷𝑊𝐿−1𝑑𝑎𝑦−1) × 𝑉𝑐𝑢𝑙𝑡𝑢𝑟𝑒 (𝐿)

𝐴𝑟𝑒𝑎 𝑒𝑥𝑝𝑜𝑠𝑒 𝑡𝑜 𝑖𝑛𝑐𝑖𝑑𝑒𝑛𝑡 𝑟𝑎𝑑𝑖𝑎𝑡𝑖𝑜𝑛 (𝑚2)

Equation 3: Determination of culture areal productivity.

where,

VP: volumetric productivity in g DW/L/day

Vculture: volume of the culture present in the cultivation system in L

3.7.5 Determination of nitrate ion concentration

The concentration of nitrate ion in the inorganic medium of Arthrospira platensis cultivation was

determined by ultraviolet absorption spectrometry, measuring the absorbance sample at 220 and 275

nm.

The measurement of the UV absorption at 220 nm allows a rapid determination of nitrate. However

dissolved organic matter can also absorb at this wavelength. Therefore, a correction was made by using

42

a second absorption value at 275 nm. At this wavelength, nitrates do not absorb, but dissolve organics

do (Equation 4).

𝐴𝑏𝑠 𝑁𝑂3− = 𝐴𝑏𝑠 (220 𝑛𝑚) − 2 𝑥 𝐴𝑏𝑠 (275 𝑛𝑚)

Equation 4: Correction of determination of nitrate ion concentration.

where,

Abs NO3-: total absorbance of nitrogen

Abs (220 nm): absorbance of nitrogen at λ=220 nm

Abs (275 nm): absorbance of nitrogen at λ=275 nm

The absorbance value was then converted to nitrate concentration using the calibration curve obtained

with the nitrate standard solutions (KNO3).

Culture samples were centrifuged at 3500 rpm for 10 minutes in the centrifuge (Hermle Z 400 K). The

supernatant obtained was diluted using fresh culture medium and HCl (1M) was added at a final

concentration of 3% (v/v) to prevent interferences from other absorbing compounds like carbonate or

hydroxide anions. Each sample was read in duplicate in quartz cuvettes, with 1 cm of path, against fresh

culture medium.

3.7.6 Pigments analysis

Chlorophylls, carotenoids and other pigments concentrations were determined by total wavelength

spectrophotometric scan of the pigment solution obtained from biomass samples by extraction with bead

beating and acetone (see Figure 3, 4 and 5 in Section 1.1.4). Each sample was read in duplicate in

quartz cuvettes, with 1 cm of path, against acetone.

After measuring the visible absorption spectrum of the pigments solution extracted from biomass, each

pigment concentration was determined by spectral decomposition: an iterative method that matched the

sum of the absorbance spectra of each accounted pigment to the measured spectrum.

The mathematical method to determinate and quantify the pigments, based on Beer-Lambert law, was

developed as a fast and inexpensive way of predicting chlorophylls and carotenoids concentration from

microalgae cultures, by A4F. The Beer-Lambert law is used to convert every absorption value in the

spectrum into a concentration of pure pigment.

A(λ)=c1ε1(λ)+c2ε2 (λ)+⋯+cnεn (λ)

Equation 5: Lambert-Beer Law.

43

where,

A(λ): Total absorbance at wavelength

ci: concentration of attenuating pigments i in the sample

εi: molar attenuation coefficient of the attenuating pigments I in the sample

As background information, it is necessary to know the pigments present in the extract under analysis,

or at least the more relevant ones, that are going to set the main tendencies of the spectrum. It is also

necessary to have the UV/vis spectrum of each pure pigment extract as well as the molar absorbance,

in order to combine all the spectra in one to reach.

3.7.7 Proteins analysis

To do the protein analysis the reference method of Lowry was used but with some modifications (Figure

25 e 26) (Lowry & Lewis, 1951).

The protein analysis allows to quantify the amount of existing protein in a given amount of dried biomass,

so it is important to do a determination of the dry weight of the same sample that undergoes the Lowry

method.

Figure 25: Aspect of the culture after addition of NaOH (1M), heating and centrifugation.

Figure 26: Final aspect of the samples that were read in spectrophotometer.

3.7.8 Elemental analysis

The elemental analysis needed to do this work were conducted by an external supplier.

44

45

Chapter 4:

Results and Discussion

To achieve the goals of this work, the experimental phase can be divided in three distinct assays that

provided the information needed. In the first assay it was possible to compare the productivities of the

different cultivation systems studied and to conclude what is the best system to grow Spirulina. The

second assay focused in the medium recirculation strategy at a pilot-scale cultivation of Arthrospira

platensis. This assay tested the culture’s productivity, the variation on the concentration of the culture’s

medium inorganic components and the impact of this methodology on cyanobacterium metabolism.

To find a different culture medium recipe that allows Spirulina growth and to verify if a formulated

seawater could be an adequate, less expensive alternative, an additional assay was done.

4.1 Assay 1: Comparison of productivity in different cultivation systems

Assay 1 was developed between March 16th and May 31st of 2016, in A4F facilities. Its goals were: to

compare the productivities between different cultivation systems and to understand what is the best

choice to cultivate the species studied.

Throughout the course of this assay some parameters measurements were done, such as optical

density, dry weight and packed cell volume. These measurements were used to calculate culture

productivity and to depict the evolution of each culture during the assay.

46

4.1.1 Productivity analysis of Arthrospira platensis in indoor flat-plate photobioreactor

The evolution of the culture present in indoor flat-plate photobioreactor in terms of concentration (g

DW.L-1) during the assay is shown in Figure 27. In this figure it is also presented the average daily

radiation (in the secondary vertical axis) during the assay. The average daily radiation data was gathered

through a weather station installed in LNEG and is presented in terms of MJ.m-2. These values of

radiation were measured outside the greenhouse.

It is very important to refer that the values of radiation inside the greenhouse can be very different from

the radiation outside - ranged between 50 to 80% lower according to time of the day and day of the year.

Unfortunately, the weather station of LNEG failed to register the average radiation between the twentieth

and last day of cultivation in this cultivation system.

The evolution of the culture present in indoor flat-plate photobioreactor in terms of volumetric productivity

(g DW.L-1 day-1) during the assay is shown in Figure 28.

Figure 27: Daily DW and average daily radiation in indoor flat-plate photobioreactor throughout the assay.

Radiation values refer to outdoor, whereas the radiation impinging on the indoor PBR can be 50-80% lower

according to the time of the day and to the day of the year.

0.0

5.0

10.0

15.0

20.0

25.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

53

63

73

83

94

04

14

2

Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

DW

(g/

L)

Days of cultivation

DW Average daily radiation

47

Figure 28: Daily volumetric productivity and average daily radiation in indoor flat-plate photobioreactor throughout

the assay. Radiation values refer to outdoor, whereas the radiation impinging on the indoor PBR can be 50-80%

lower according to the time of the day and to the day of the year.

From the analysis of Figures 27 and 28 it is possible to see that the dry weight of the culture ranged

between 0.07 and 1.6 g/L. This maximum was obtained on 30th day of cultivation. Between the 1st and

the 41st days of cultivation, the average volumetric productivity was 0.05 g/L/day. The maximum value

of volumetric productivity was obtained on 27th and 28th days of cultivation.

Regarding Figure 27, it is possible to observe that, in two occasions, the concentration dropped

drastically during the assay. This abrupt fall of concentration symbolizes the two renewals of the

cultures. These renewals are essential to maintain the culture in the linear growth phase during the

assay.

By observing Figure 28, the influence of incident radiation on culture productivity is not very clear. The

variation in daily productivity goes along with variation in average daily radiation for 19 part of the days,

however for some days the variation in daily productivity shows an opposite tendency of variation in

average daily radiation. These variations are not necessarily proportional between them due to other

factors impacting on culture productivity, such as temperature, pH, culture concentration and

contaminants.

As microalgae cells are biological systems and, therefore, have a detectable response time to

environment changes, it is normal to sometimes observe a delay between the variations in culture

productivity and average daily radiation: there was a decrease in volumetric productivity between the 1st

and 3rd days and between the 6th and the 7th days of cultivation that can be explained by this fact. Apart

0.0

5.0

10.0

15.0

20.0

25.0

-0.03

0.00

0.03

0.06

0.09

0.12

0.15

0.18

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

53

63

73

83

94

04

14

2

Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

Vo

lum

etri

c P

rod

uct

ivit

y (g

/L/d

ay)

Days of cultivation

Volumetric Productivity Average daily radiation

48

from that, on the 6th day of culture a renewal of 83% was done. This renewal also contributed to a

decrease in volumetric productivity.

Two points were eliminated (2nd and 20th days) from the graphics. With these points in the graphics it

was possible to observe the existence of spikes and “valleys” for days in a row. Inclusively, one of the

days whose growth doubled the average was followed by one of no growth. This fact results of probable

errors in sampling or in determination of DW. The error ranges easily corroborated that the cause was

the experimental error.

To well understand the influence of average daily radiation in volumetric productivity of the culture,

missing average radiation data are essential.

4.1.2 Productivity analysis of Arthrospira platensis in cascade raceway

The evolution of the culture present in cascade raceway in terms of concentration (g DW.L-1) during the

assay done is shown in Figure 29. In this figure is also shown the average daily radiation (in the

secondary vertical axis) during the assay.

The evolution of the culture present in cascade raceway in terms of volumetric productivity (g DW.L-1

day-1) during the assay is shown in Figure 30.

It is important to refer that the first assay done in this cultivation system failed, so only the second assay

was used to calculate and compare productivities.

The weather station of LNEG failed to register the average radiation for the first day of this culture.

Figure 29: Daily DW and average daily radiation in cascade raceway throughout the 2nd assay.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 1 2 3 4 5 6 7 8 9 Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

DW

(g/

L)

Days of cultivation

DW Average daily radiation

49

Figure 30: Daily volumetric productivity and average daily radiation in cascade raceway throughout the 2nd assay.

After inoculation the culture started its growth attaining after 8 days the DW of 0.79 g/L. The average

volumetric productivity of the culture was 0.08 g/L/day.

Regarding Figure 29, it is easy to remark that he culture grew very slowly during the assay.

Observing Figure 30, it is possible to verify that incident radiation on culture was practical constant

during the assay as well the culture growth.

The major increase of volumetric productivity occurred near of the end of the assay.

After reviewing further available data from the culture, it was possible to identify and verify that the 6 th

day of the culture was a Monday. On this day nutrients were supplemented to the culture. Bearing in

mind that the culture did not receive any nutrients for the previous week, it is probable that some

micronutrients essential to cyanobacterium growth did not exist in the medium. When the culture

received the missing nutrients, the productivity restarted to increase once more.

On 8th day of cultivation, the biomass of the cultivation system was harvested and dried in a spray-dryer.

Due to operational constraints it was not possible to extend this assay or repeat it. Future research

should include the test of productivity in this system with a pump frequency higher than the one used in

this assay.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 1 2 3 4 5 6 7 8 9 Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

Vo

lum

etri

c p

rod

uct

ivit

y (g

/L/d

ay)

Days of cultivation

Volumetric productivity Average daily radiation

50

4.1.3 Productivity analysis of Arthrospira platensis in outdoor flat-plate photobioreactor

The evolution of the culture present in the outdoor flat-plate photobioreactor in terms of concentration

(g DW.L-1) during the assay done is shown in Figure 31. In this figure, it is also shown the average daily

radiation (in the secondary vertical axis) during the assay.

Unfortunately, the weather station of LNEG failed to register the average radiation between the start and

the thirteenth day of cultivation in this cultivation system.

The evolution of the culture present in the outdoor flat-plate photobioreactor in terms of volumetric

productivity (g DW.L-1 day-1) during the assay is shown in Figure 32.

Figure 31: Daily DW and average daily radiation in outdoor flat-plate photobioreactor throughout the assay.

Figure 32: Daily volumetric productivity and average daily radiation in outdoor flat-plate photobioreactor

throughout the assay.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

5 Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

DW

(g/

L)

Days of cultivation

DW Average daily radiation

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

-0.30

0.00

0.30

0.60

0.90

1.20

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

5 Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

Vo

lum

etri

c P

rod

uct

ivit

y (g

/L/d

ay)

Days of cultivation

Volumetric productivity Average daily radiation

51

From the analysis of Figures 31 and 32 it is possible to verify that the dry weight of the culture ranged

between 0.32 and 2.8 g/L. After inoculation, the culture started to grow until 14th day. In this day the

culture entered at stationary stage.

Figure 31 highlights a renewal of 30% of this culture. The concentration dropped on 20th day of

cultivation, when the renewal was done to maintain the culture in linear growth phase

Throughout the assay the average volumetric productivity was 0.09 g/L/day.

The presence of contaminants in the culture can lead to the decrease of the observed volumetric

productivity of A. platensis.

It is also important mentioning that the number of foreign organisms in the cultures is directly proportional

to the culture handling operations and addition (involving the opening of the reactors) and also to the

days of cultivation. On 20th day of cultivation a culture renewal was done. This process was probably

the origin of contaminations.

4.1.4 Productivity analysis of Arthrospira platensis in conventional raceway

The evolution of the culture present in conventional raceway in terms of concentration (g DW.L-1) during

the assay done is shown in Figure 33. In this figure is also shown the average daily radiation (in the

secondary vertical axis) during the assay.

The evolution of the culture present in raceway in terms of volumetric productivity (g DW.L-1 day-1) during

the assay is shown in Figure 34.

Figure 33: Daily DW and average daily radiation in conventional raceway throughout the assay.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 1 2 3 4 5 6 7 8 9 10 11 12 Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

DW

(g/

L)

Days of cultivation

DW Average daily radiation

52

Figure 34: Daily volumetric productivity and average daily radiation in conventional raceway throughout the assay.

From the analysis of Figures 33 and 34 it is possible to see that the dry weight of the culture ranged

between 0.36 and 0.85 g/L. During the assay, the culture grew very slowly and the maximum dry weight

was obtained on 8th day of cultivation. The average volumetric productivity between 1st and 11th days of

cultivation was 0.03 g/L/day.

Figure 34 suggests that incident radiation does not affect the culture productivity very much.

Because this system is an open system, the risk of contaminations is higher. During this assay it was

possible to observe different contamination agents: bacteria, filamentous bacteria and others such as

pollen (which is innocuous) (Figure 35). However, the volumetric productivity remained constant despite

the high contamination level.

Figure 35: Contaminant (pollen) observed by microscopic observation of a sample of conventional raceway

(magnification 40x).

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

-0.09

-0.06

-0.03

0.00

0.03

0.06

0.09

0.12

0.15

0.18

0.21

0 1 2 3 4 5 6 7 8 9 10 11 12

Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

Vo

lum

etri

c P

rod

uct

ivit

y (g

/L/d

ay)

Days of cultivation

Volumetric productivity Average daily radiation

53

4.1.5 Comparison between productivities analysis

In order to compare if different cultivation conditions have impact on the Spirulina’s metabolism, a

pigment and protein analysis of samples (culture and powder from CRW) of the different cultivation was

done. It was possible to observe that independently of the conditions of each system, the percentage of

protein was maintained in a range between 60 and 70%, which, as explained in section 1.1.4, is the

normal value. In Table 9 are summarized the values of protein analysis, the most important factors that

can affect the productivity and the average productivities.

To determine the areal productivities for each system an Equation 3 was used. Afterwards, an average

of all the values of areal productivity was done. These values are also shown in Table 9.

54

Table 11: Summary of factors that have impact in productivity. Productivities of the different cultivation systems under study.

Cultivation

System

Months of

Cultivation

% of

Protein

Average

Temperature

(ºC)

Minimum

Temperature

registered

(ºC) *

Average

pH

Average

Radiation

( MJ/m2)

Average

Dry

Weight

(g/L)

Average

Volumetric

Productivity

(g/L/day)

Cultivation

system’s

photosynthetic

area (m2)

Average Areal

Productivity

(g/m2/day)

Indoor flat-plate

photobioreactor

March and

April

61 %

(5/4/16) 22.01 16.70 9.56 14.98 0.55 0.05 0.772 4.3

Cascade

raceway

April and

May

60%

(3/5/16) 24.24 13.30 9.90 29.13 0.42 0.08 3.73 6.5

Outdoor flat-

plate

photobioreactor

April and

May

62%

(3/5/16) 21.55 14.60 9.88 21.13 1.6 0.09 0.570 9.6

Conventional

Raceway

May and

June - 21.49 12.30 9.63 25.90 0.63 0.03 5.00 3.8

*This temperature can be lower since it was not possible to monitor the cultivation temperature during the night. This variation can be more significant in

outdoor cultivations.

55

To compare the productivities between the different cultivations systems used it is preferable to use the

AP, because this productivity is independent of the volume of the culture present in the system.

To better understand the impact of this study for further knowledge on this subject, a research of typical

values of areal productivities for several systems was done.

Table 10 compares the values between the average areal productivities obtained in this project and

typical average areal productivities available in literature.

Table 12: Comparison between average areal productivity obtained and the average areal productivity present in literature

Cultivation

system

Average

Radiation

of the

assays

(MJ/m2)

Average

Areal

Productivity

(g/m2/day)

obtained

Comments

Average

Radiation

of

literature

(MJ/m2)

Average

Areal

Productivity

(g/m2/day)

of literature

Reference

Outdoor flat-

plate

photobioreactor

21.1 9.6 - 25.5 17.4

(Vonshak,

2002)

Indoor flat-plate

photobioreactor 15.0a 4.3 -

Not

available

Not

available

(Vonshak,

2002)

Cascade

raceway 29.1 6.5

Possible

shear

stress by

pumping.

Duration of

cultivation

of few days

Not

available 18

(Borowitza &

Moheimani,

2013; Ojamäe,

2011)

Conventional

Raceway 25.9 3.8b

Poor

Mixing

Not

available 15

(Vonshak,

2002)

a Affected by a reduction between 50 and 80% in relation to the outdoor flat-plate photobioreactor due to the greenhouse.

b The data were obtained before being performed any culture medium recirculation.

Table 10 shows that the average areal productivities available in literature is higher than the average

areal productivities obtained. As there was not available values for indoor flat-plate PBR, these values

were calculated. Starting from the literature values of outdoor flat-plate PBR and assuming the same

56

assumptions used in this thesis in terms of shade caused by greenhouse, the radiation would vary

between 5.1 to 12.8 MJ/m2.

In this cause and assuming that the incident radiation is the only limiting factor, the areal productivity

estimated would be 3.5- 8.7 g/m2/day, which is in agreement with the experimental result obtained. It is

worth mentioning that the values verified on indoor flat-plate, outdoor flat-plate and raceway result from

cultivations under climatic conditions of Florence. It is also important referring that the values obtained

on flat-plate systems are values of cultivations that occurred during the summer without any inclination

of the system.

Just because of the place where the different projects were held, the incident radiation that reaches the

cultivation systems is also different which may explain the observed differences in productivities. On the

other hand, the fact that the project in Florence has occurred in the summer is synonymous of a greater

amount of sun exposure which directly affects the photosynthesis and can lead higher temperatures.

This last fact is most important in outdoor systems where the control of temperature can be inexistent

and where the fluctuations in temperature of day and night can be high causing cell damage.

Higher temperatures usually mean higher respiration and respiration at night results in loss of biomass,

although higher night respiration at 25ºC than at 35ºC was observed in Arthrospira platensis (Torzillo,

Sacchi, Materassi, & Richmond, 1991). This is probably related to the fact that 25ºC is suboptimal for

this species. It is important to stress that 25ºC was the set—point defined for the flat-plate

photobioreactors cultivation systems. In this way, an increase of areal productivity would be expected if

the assay was carried out at 35ºC. These facts may justify the significant discrepancy between both

values.

Table 10 sets forth that the cultivation system which presents the higher average AP is the outdoor flat-

plate photobioreactor whilst the raceway is the cultivation system with lower average AP.

It is curious to verify that the cultivation systems present the higher and the lower AP are both outdoor

cultivation systems.

So why outdoor cultivation systems present such a different AP? Are there conditions that can cause

this phenomena? What are these conditions?

First of all, it is important remembering that flat-plate photobioreactor is a closed system and

conventional raceway is an open race. Thus, the culture present in conventional raceway was more

vulnerable to external conditions, such as: contaminations, evaporation, rainfall and others. On the other

hand, it is also very important to remember that the conditions of temperature regulation and CO2 supply

were different in the two systems.

In flat-plate photobioreactor there was a temperature control (set-point of 25ºC) whereas in the RW the

temperature was controlled by evaporation. This fact can allow a smaller thermal amplitude within flat-

plate photobioreactor between day and night, while in RW the thermal amplitude can be quite high. This

temperature range may be the main reason for the lower productivity observed in RW.

57

Relatively to CO2 supply, this only existed in flat-plate photobioreactor being inexistent in the RW. This

means that the culture present in RW established exchanges with the atmospheric air using CO2 present

to do photosynthesis. The high pH existing in the RW contributed to improve the absorption of the air’s

CO2 (Jourdan, 2006).

In the addition of the pH of the culture also the string presents a major role in the absorption of CO2.

While the flat-plate photobioreactor has a diffuser that ensures a good homogenization of the culture, in

the RW only the paddle wheel contributes to the unrest, which may not be sufficient for an ideal CO2

absorption.

It is quite probable that the AP of RW will increase if CO2 from another source is injected in the culture

and if the paddle wheel frequency increases too. Despite the pH contribute to the increase of efficiency

of gas exchange, it is noteworthy to point out that the mixing of the culture also plays a major role.

The AP values obtained for the two other cultivation systems are expected when compared with the

values obtained for outdoor flat-plate photobioreactor and RW. It makes perfect sense that the indoor

flat-plate photobioreactor presents an AP of 4.3 g/m2/day because, despite being a system with greater

control of parameters (regulation of temperature, injection of carbon dioxide, being a closed system,

etc.…), it receives less radiation inside the greenhouse.

In general, the values obtained for the different cultivation systems under study are quite reasonable if

we take into account the conditions the assays were conducted and, particularly, the year’s season.

To conclude, the cultivation system that proved to be more effective to cultivate A. platensis was the

outdoor flat-plate photobioreactor. However, it is quite likely that with more control over the two other

outdoor systems and assays conducted during the summer, the results would be much closer to those

of the literature.

On the other hand, it is important to bear in mind the benefit-cost (construction, reagents, water, energy

consumption and others) ratio of areal productivity when we decide to choose a cultivation system to

grow Spirulina.

To do a detailed analysis of costs a list of all the expenses made for each assay carried out in the various

systems would be required. The total sum expended with the AP obtained for each would be the decider.

In this way it is possible that the system with greater productivity and less cost (ideal situation) may be

other than the outdoor flat-plate photobioreactor.

58

4.2 Assay 2: Analysis of a medium recirculation strategy of Arthrospira platensis production at a pilot-

scale in a conventional raceway

Assay 2 was developed between May 19th and June 24th of 2016, in A4F facilities. The purpose of the

study was twofold; first, to verify if an Arthrospira platensis culture could be maintained in good

productivity conditions in a cultivation system where the culture medium was being recirculated; and

second, whether the nutritive medium formulation used at the time was indicated for this cultivation

system.

Throughout the course of this assay, dry weight measurements were taken from the Spirulina culture of

raceway with 670 L, whose culture medium was recirculated according to resources availability and

production needs.

These measurements were used to calculate culture productivity in each period of time between two

renewals - renewal cycle - and depict the evolution of the culture during the assay.

To identify possible productivity changes caused by incident radiation, this parameter was also taken

into account.

At the end of the assay, a culture sample was collected. This sample and the media elemental

composition was assessed and compared to the culture medium recipe to examine the nutritive medium

suitability for conventional raceway cultivation of Spirulina.

59

4.2.1 Productivity analysis of Arthrospira platensis culture using recycled culture medium

Throughout this assay, after inoculation, three renewal cycles with different duration were examined, as

the decision of harvesting a culture was made according to culture evolution and state. In Table 11 the

renewal cycles which were analysed in this assay are summarized

Table 13: Renewal cycles of conventional raceway

Starting

date

Renewal

date

Renewal

cycle

Production

time (days)

Renewal

rate (%)

Recirculation

rate (%)

Average

Cycle

Temperature

(ºC)

19/05 31/05

(12th day) 0 12 54 83 21.5

01/06 07/06

(19th day) 1 6 54 83 24.2

08/06 16/06

(28th day) 2 8 50 63 22.7

17/06 24/06 3 7 - - 25.0

By observing Table 11, it can be verified that the 2rd renewal cycle had a lower renewal rate and the

amount of medium that returned to the RW was lower (lower recirculation rate) too.

To understand and evaluate the influence of culture medium recirculation in culture productivity, it is

essential to study the evolution of the productivity during the time of the assay.

The dry weight should be analysed in parallel with the culture productivity, since its evolution during the

assay can be useful to trace factors that can affect culture growth. On the other hand, the incident

radiation is another factor that should also be analysed because it can have a great influence on culture

growth.

Figure 36 shows the dry-weight alongside the average daily radiation for each cultivation day in raceway,

while Figure 37 shows the volumetric productivity alongside the average daily radiation.

Figure 38 shows the dry-weight alongside the volumetric productivity.

During the assay, culture temperature and pH remained within acceptable range for growing A.

platensis. A standard production procedure that included microscopic observations and nitrate

60

concentration measurements was applied to the culture, enabling the detection of anomalies which

could have impact on culture growth.

Figure 36: Daily DW and average daily radiation in conventional raceway throughout the assay.

Figure 37: Daily volumetric productivity and average daily radiation in conventional raceway throughout the assay.

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

-0.15

-0.10

-0.05

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

53

63

7

Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

Vo

lum

etri

c p

rod

uct

ivit

y (g

/L/d

ay)

Days of cultivation

Volumetric productivity Average daily radiation

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

53

63

7

Ave

rage

dai

ly r

adia

tio

n (

MJ/

m2

)

DW

(g/

L)

Days of cultivation

DW Average daily radiation

61

Figure 38: DW and daily volumetric productivity in conventional raceway throughout the assay.

By observing Figures 36 and 37, it becomes clear that the variation of DW and volumetric productivity

are not influenced by incident radiation.

From Figure 36 analysis, it is possible to see that the dry weight of the culture was in a range between

0.27 and 1.01 g/L. The maximum value was obtained on 35th day of cultivation.

The maximum value of volumetric productivity was obtained at the same day of cultivation of the

maximum DW value noticed in Figure 36.

In Figure 36 it is also possible to observe that, in 3 occasions (12th, 19th and 28th days of cultivation), the

concentration dropped drastically during the assay. These three abrupt falls of concentration symbolize

the three renewals and the recirculation of culture medium. The major drop of VP occurred between the

27th and the 29th days of cultivation. In addition to the renewal that occurred during this period of time

and after checking other available data, it was possible to see that in these particular days the

temperatures were lower than the average temperature registered. This can lead to the loss of VP that

happened.

Altogether, from the analysis of Figure 38, it is possible to say that DW and VP follow the same growth

trend.

Bearing in mind that the assay was held in an open pond, as mentioned already, which does not allow

a total control over all culture parameters or monitoring all the details of operations performed to the

culture, all renewal cycles were in fact very similar to each other.

Analysing Figure 36 or Figure 38 it can be seen that there is several times, after renewals, a decline of

DW. These facts may correspond to an experimental error that can have two different explanations/

origins.

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

0 1 2 3 4 5 6 7 8 91

01

11

21

31

41

51

61

71

81

92

02

12

22

32

42

52

62

72

82

93

03

13

23

33

43

53

63

7

Vo

lum

etri

c p

rod

uct

ivit

y (g

/L/d

ay)

DW

(g/

L)

Days of cultivation

DW Volumetric productivity

62

At the moment when the sample was collected the culture was not completely homogenized. This fact

may be the probable cause of the phenomenon observed, since RWs have a poor mixing regime

originating in the paddlewheel. As such, the first data point for each cycle was discarded to perform the

calculations that follow.

In conclusion, all the data and results suggest that medium recirculation did not impact negatively on

the culture volumetric productivity.

Finally, to verify if the productivity depended of the incident radiation, two graphs were compared

(Figures 39 and 40). Figure 39 shows the average areal productivity for each renewal cycle, while

Figure 40 shows the average areal productivity per unit of incident radiation for each renewal cycle in

conventional raceway. The comparison between the two graphs shows that the points of each cycle

have the same tendency. This fact suggests that the culture productivity was independent of the

radiation in each cycle, which was fairly constant and with high average values, especially since the

average values of culture concentration were low (so radiation would not be a limiting growth factor in

the conditions tested).

Figure 39: Average areal productivity in each renewal cycle in conventional raceway.

Figure 40: Average areal productivity per unit of incident radiation in each renewal cycle in conventional raceway.

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

0 1 2 3 4

Ave

rage

Are

al P

rod

uct

ivit

y (g

/m2

/day

)

Renewal cycle

0.00

0.10

0.20

0.30

0.40

0.50

0 1 2 3 4

Pro

du

ctiv

ity

/Rad

iati

on

(g/m

2/d

ay)/

(MJ/

m2

)

Renewal cycle

63

The results of the Figure 39 show that the AP remained constant throughout the assay with the exception

of the cycle 0 that presents a lower productivity. To better understand which were the factors that had

influence in the obtained results, a graph (Figure 41) based on the values of the different parameters of

each renewal cycle (Table 12) was drawn.

Table 14: Summary of some parameters that can influence the culture productivity of each renewal cycle.

Renewal

cycle

Average AP

(g/m2/day)

Average

DW (g/L)

Specific

growth rate

(µ) (day-1)

Average

Temperature

(ºC)

Average

Radiation

(MJ/m2)

0 3.6

0.64 0.06 21.5 23.9

1 6.6

0.54 0.17 24.2 25.7

2 8.7

0.54 0.21 22.7 25.9

3 6.8

0.77 0.15 25.0 28.6

Figure 41: Comparison of the different factors that can influencing the culture productivity in each renewal cycle.

Average radiation in MJ/m2 and Specific growth rate in day-1.

23.9 25.7 25.9 28.6

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

0 1 2 3

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Ave

rage

Are

al P

rod

uct

ivit

y (g

/m2

/day

)

Renewal cycle

Ave

rage

DW

(g/

L)

Average Radiation Average Areal Productivity Average DW Specific growth

64

The results of the Figure 41 can corroborate that the cycle 0 presents a lower productivity while the last

three cycles are very similar to each other. The different parameters were maintained approximately

constant for these three cycles. It is possible to remark that the culture productivity increased when the

first recirculation was performed. The fact that the average AP and specific growth rate have the same

constant progress in consecutive renewal cycles evidences that the culture kept the same overall

healthy condition for the same cycles.

This finding concurs with the hypothesis of culture growth not being limited by recirculation of the culture

medium.

In this way and against the expected results, the recirculation strategy helps the cultivation. However it

is very important to stress that sometimes in the start of cultivations (after inoculation), there is an

adaption period of the cells that can negatively affect the areal productivity and justify the results

observed for the cycle 0.

It would be relevant to consolidate all these results with a new assay with the same conditions.

In a similar way to what was done in the comparison study of productivities between different cultivation

systems, so did the recirculation study of the medium became a monitoring of the amount of proteins

present in the cells.

At the end of each renewal cycle and before a new renewal cycle a protein analysis was performed.

The results of these analyses are summarized in Table 13, below.

Table 15: Results of protein analysis of each renewal cycle of conventional raceway

Date % of Protein

07/06

(between 1st and 2nd cycles) 66

16/06

(between 2nd and 3rd cycles) 64

24/06

(end of 3rd cycle) 67

According to the results shown in this section, evidences support the possibility to cultivate Arthrospira

platensis in a cultivation system with recirculation of the culture medium, maintaining the normal amount

of proteins in biomass.

65

4.2.2 Elemental analysis of fresh and recycled culture medium

Firstly, to perform the comparison between the fresh sample and recycled medium sample collected at

the final of 3rd renewal cycle, to the elemental composition corresponding to the fresh culture medium,

the elemental concentrations of the nutritive medium were added, according to the nitrate concentration

obtained from the elemental composition of the recycled medium (see Table 7 in section 3.5). This

methodology allows compositions to be comparable.

These are the values that would be expected to be observed in the recycled medium sample if there

were not more inputs to the cultivation system, if there were not any outputs and if the microalgae did

not produce or consume nutrients. Thus, these values become the most suitable to compare with the

values of the elemental analysis obtained for the recycled medium sample.

The variation between fresh medium and recycled medium is shown in Table 14.

Table 16: Percentage of concentration variation between fresh medium and recycled medium. Only variations which were equal or superior to 30% in absolute value were considered relevant and therefore specified.

Element Δ (%)

Bicarbonate -31,29

Chloride -67,39

B -60,50

Ca 458,86

Cu 36,65

Fe -89,66

Mg -92,19

Mn 43,52

Mo 81,58

Na -30,61

P 31,01

Zn -78,35

Co 27,67

Only elements which are considered relevant for microalgae production were taken into account in this

analysis. Also, only variations above of 30% in absolute value were considered relevant, and therefore

pointed out.

Comparing to the composition that was expected (fresh medium + nutritive medium), the recycled

medium had an excessive concentration of calcium, molybdenum and manganese and a slight excess

of phosphorous, copper and cobalt. These elements suffered a positive variation, so it suggests that the

first elements were accumulated faster than elements such as phosphorous, copper or cobalt in the

recycled medium.

66

The variations between calcium, molybdenum and manganese concentrations in the fresh medium and

recycled media were, respectively, five-, two- and one- fold higher in the recycled medium. On the other

hand, the recycled medium had a little shortage of sodium, bicarbonate and chloride. Boron, zinc, iron

and magnesium were being depleted faster than the other elements that present a negative variation.

In fact, magnesium, iron and zinc were practically exhausted from the recycled medium, meaning that

these elements could be regarded as limiting nutrients in this cultivation. The variations between

magnesium, iron and zinc were thirteen-, nine- and two- times lower in the recycled medium,

respectively.

Calcium is the element that presents the major positive variation between fresh and recycled medium.

This fact can be explained considering calcium’s characteristics – it is an element that can easily

precipitate in the culture medium when replaced of water volume evaporated was done by tap water.

This water source contains a considerable amount of calcium in its composition, and the culture received

calcium only from this source.

The calcium can precipitate in the form of Ca3(PO4)2 or in the form of CaCO3 during daily additions of

freshwater to compensate the evaporation losses. This phenomenon results in the reduction of alkalinity

and to a certain extent loss of iron from the system (Vonshak, 2002).

Calcium possesses an important structural role in cyanobacteria. Throughout this assay no structural

problem was seen under microscope observation. Hence, the results suggest that Arthrospira platensis

culture consumed little and just the necessary amount of calcium and that its presence in the recycled

medium sample is due to accumulations during the assay.

The calcium accumulations can be in the form of the exopolysaccharide Calcium-Spirulan (see section

1.1.4) since it was possible to observe a polysaccharide on the bottom of the RW after one week and a

half from the date of recycled sample collecting (Belay, 2002; Hayashi & Hayashi, 1996; Pinotti &

Camilios Neto, 2004).

Concerning molybdenum (this element is used to help nitrogen absorption), its concentration in fresh

and recycled medium is not very high, however it is possible to observe a significant positive variation

between both concentrations. Taking into account that the amount of molybdenum is low in the nutritive

medium, this variation seems to have been caused by a low consumption of this element by the culture,

which can lead to the accumulation observed.

To better understand how the accumulation of the elements with positive variations occurred throughout

the assay and according with each renewal cycle, it would be necessary to perform a set of elemental

analyses of samples of recirculated medium at the end of each cycle.

With the results obtained in mind, there are some changes that could be done in the nutritive medium

recipe in order to optimize it for cultivation systems with recycling of the culture medium:

Because magnesium, iron and zinc are important macronutrients and micronutrients,

respectively, for Spirulina and given that these elements could be regarded as limiting nutrients,

67

their concentrations in the recipe should be increased. Magnesium is a fundamental

macronutrient used for chlorophyll production. The fate of iron in the alkaline medium of

Spirulina culture is poorly understood, but it can be associated to the production of cytochromes

(Vonshak, 2002). Zinc also has an important role in metabolic processes taking place in cells

(Richmond, 2004).

Calcium, molybdenum, copper, phosphorous and cobalt were accumulating in the medium.

Even if none of them exceed a concentration value which has been reported as toxic for some

microalgae, all the elements should have their concentrations reduced in the medium, especially

the calcium and the molybdenum. To decrease the amount of calcium the use of another water

source (softer) is suggested. To decrease the amount of the rest of the elements a direct

reduction in the nutritive medium is suggested.

This strategy has the objective of avoiding future toxic accumulation of any of them in the culture

medium and also generate savings in the culture medium costs, since nutrients costs can

correspond to 15-25 percent of the total production costs (Vonshak, 2002).

Actually detailed knowledge of the nutrient uptake kinetics of Spirulina in pilot and large-scale open

ponds systems and the fate of certain nutrients in the high pH of the medium is lacking. When such

information is available, it will help to minimize nutrient costs or increase productivity, without a

doubt.

Of the analysis and comparison between the elemental compositions of fresh and recycled medium

during this project, a reformulation of the nutritive medium is suggested to fit the needs of a raceway

with medium recirculation, as displayed in Table 15.

To sum up, it is possible to say that it is possible to cultivate Arthrospira platensis at a pilot-scale in

a raceway with medium recirculation without productivity loss during at least 36 days, if the medium

described in Table 7 is used.

With the adequate alterations to the nutritive medium and water used an increase of cultivation

period maintaining at least the same productivity is expected. However, an increase of productivity

can be anticipated as limiting nutrients become more available for microalgae growth.

68

Table 17: Suggestion of reformulation of nutritive medium put forward in Table 7.Comparison between the reference recipe developed by A4F and the suggestion of nutritive medium according to the assay performed.

Reagent Δ (%)

NaNO3 [+ 0]

Nutritive

medium

KH2PO4 [+ 0]

Disodium EDTA [+ 125]

MgSO4·7H2O [+ 100]

FeSO4·7H2O [+ 100]

ZnSO4·7H2O [+ 50]

MnCl2·4H2O [- 25]

Na2MoO4·2H2O [- 50 ]

CoCl2·6H2O [- 15]

CuSO4·5H2O [- 25]

An elemental analysis of biomass should be performed with the objective of closing the mass balance.

A carbon balance would be also interesting to perform, however there are some constraints such as the

fact that the reactor is open to air.

To better understand the economic impacts of the strategy of recycling of culture medium and to

understand how much it is possible to save using this strategy, a detailed economic analysis to the

whole process should be made.

However, the results obtained point to an effective reduction of the costs despite of a small difference

of productivity between renewal cycles.

69

4.3 Assay 3: Analysis of Arthrospira platensis growth in alternative culture medium

As previously mentioned, an additional assay was done with the purpose of finding a different culture

medium recipe that allows Spirulina growth.

Seawater is the best alternative to grow Spirulina because this medium reduces the consumption of

water and chemicals for the formulation of the culture medium, such as the bicarbonate concentration.

Therefore, seawater is a cheap medium for the mass cultivation of Spirulina, especially if there is

evidence that productivity values will be steadily maintained (Jourdan, 2006; Leema, Kirubagaran,

Vinithkumar, Dheenan, & Karthikayulu, 2010; Materassi, Tredici, & Balloni, 1984; Tomaselli et al., 1987;

Tredici, Papuzzo, & Tomaselli, 1986).

On the other hand, this new culture medium allows the cultivation of Spirulina in several tropical arid

areas where climatic conditions are favourable for the development of this cyanobacterium but

freshwater is scarce (Materassi et al., 1984; Tomaselli et al., 1987).

As a result, a comparison between using fresh culture medium and formulated seawater as culture

medium was made.

Firstly, a gradual adaptation of Spirulina to the salinity was promoted to avoid a cell osmotic shock.

To accomplish this intention, sodium chloride was added to the culture medium recipe presented in

Table 7 in a concentration of 5 g/L in a total volume of 2L. As it is possible to understand by observing

Figure 42, the Spirulina presented a very similar growth when compared to the control (0 g/L). It is, then,

possible to argue that A. platensis adapted easily to a culture medium with 5 g/L of sodium chloride.

With the cells of cyanobacterium adapted to 5 g/L, an increase of salinity to 7.5 g/L was promoted. The

results of Figure 43 show that A. platensis also has the capacity to adapt to this salinity.

After that, weekly, a more practical scale-up methodology was used to continuous increase salinity to

10, 15 and 20 g/L, by renewing around 90% of the culture volume with increased salinity. OD

measurements were discarded due to the formation of agglomerates with the increase of salinity, before

the adaptation is concluded in each step up to the salinity value of 20 g/L, the cells took a longer time

to respond to the culture medium and grow.

70

Figure 42: Evolution of the optical density (630 nm) of Arthrospira platensis in a culture medium (control) and in a

culture medium with 5 g/L of sodium chloride.

Figure 43: Evolution of the optical density (630 nm) of Arthrospira platensis in a culture medium (control) and in a

culture medium with 7.5 g/L of sodium chloride.

After being sure that the cells were completely adapted to a salinity of 20 g/L, the process was repeated

in tap water with sodium chloride at 30 g/L. This process tested if Spirulina could grow in tap water with

sodium chloride and nutritive medium only.

It can be concluded that although Spirulina is not a marine organism, the species used in this assay

adapted easily to formulated seawater (30 g/L). However, several physiological aspects of the growth

of Spirulina in formulated seawater remain to be clarified in order to derive the best benefits from this

opportunity.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 1 2 3 4 5 6 7 8 9 10

Days of cultivation

OD ( 0 g/L) OD ( 5 g/L)

0

0.1

0.2

0.3

0.4

0.5

0.6

0 1 2 3 4 5 6 7 8 9

Days of cultivation

OD ( 0 g/L) OD ( 7,5 g/L)

71

Chapter 5:

Conclusions and Future Work

Regarding the comparison of productivities in the different cultivation systems studied, this research

suggests that it is possible to cultivate Arthrospira platensis in all cultivation systems tested – indoor and

outdoor flat-plate photobioreactors; cascade raceway and conventional raceway- with reasonable

values of productivity – between 3.8 and 9.6 g/m2/ day.

It was also possible to conclude that the cultivation system which allowed a higher productivity was the

outdoor flat-plate photobioreactor, while the cultivation system that presented the lower productivity rate

was the conventional raceway, probably due to the poorer mixing associated with this type of system.

When comparing the productivity of all cultivations to the literature values, the values obtained in this

work were significantly lower, and one very plausible explanation is the lower temperature of these

cultivations. All systems can have their values of productivity increased if the assays are made during

the summer. The culture growth depends of the incident radiation and temperature and in this season

the average daily incident radiation and temperature are higher. If the control over cultivation parameters

also increases, for example artificially increasing the temperature of the flat-plate photobioreactors, the

productivity in these systems can be increased too.

Productivity is one of the major factors to be taken into account when choosing a cultivation system, yet

all costs associated to the system also have to be taken into account. The balance between productivity

and costs is very important to make the right choice and obtain the maximum profit possible. In this way,

the best choice could even be the cultivation system with lower productivity. Nevertheless, in this project

it was not possible to reach a stage to perform an economical study to verify which is the best productivity

vs costs option.

Regarding medium recirculation, this study indicates that it is possible to cultivate Arthrospira platensis

in a pilot-scale cultivation system, especially in conventional raceways, with medium recirculation for at

least 36 days with recirculation rates between 63 and 83%.

Despite the high values in the renewal and recirculation rates, the results lead to the conclusion that for

Spirulina cultures there were no losses in culture productivity due to medium recirculation as performed

in this work.

Results of this assay show that some elements were lacking or in excess in the recycled medium, which

indicate that these elements were being added to the culture medium through nutrients or make-up

water in an unbalanced proportion when comparing to their consumption rates by the culture of A.

platensis.

72

Therefore, and to avoid toxic effects and future nutrient constraints, it is recommended the use of a

softer water to replace the water volume evaporated and it is also recommended to perform some

adjustments in the nutritive medium recipe.

This conclusion led to the optimization of the nutritive medium recipe in cultures of Spirulina platensis.

Concerning the assay of alternative culture medium to growth A. platensis it is possible conclude that

the cells of this cyanobacteria could adapt and grow in a formulated seawater (30 g/L).

It is worth mentioning that further research and development of the growth of Spirulina in formulated

seawater could contribute significantly to improving the economic perspectives of this unconventional

protein source. At the same time its industrial exploitation in many warm countries could become more

disseminated.

With the end of this project, it becomes evident that there is still much work to be done to better

understand the behaviour of Arthrospira platensis.

Future research should include some assays that are described below:

New test of productivity in CRW cultivation system with a pump frequency higher than the one

used in this project (19 Hz);

Test of productivity in a tubular photobioreactor (PBR);

Test of productivity in flat-plate photobioreactors and others with a higher temperature set-point

(35 ºC)/ heating;

Productivity test at pilot scale using tap water and sodium chloride (30 g/L) as culture medium

and using flat-plate photobioreactors as cultivation system. Compare the results with the results

obtained in this project;

New assay of medium recirculation of Arthrospira platensis production in conventional raceway,

using the recipe obtained with this project as culture medium;

Test in the laboratory (controlled radiation conditions) to compare the productivity using

optimized medium fromA4F and the productivity with the medium published in the literature

(where benchmark productivity data is published).

73

Chapter 6:

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