Recycling of the culture media for pilot scale production of Arthrospira platensis ... · Recycling...
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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
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In memory of one of the great men of my life. Thank you for all.
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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
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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
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Chapter 6: .................................................................................................................... 73
Bibliography ................................................................................................................ 73
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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
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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
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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
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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
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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
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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.
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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.
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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).
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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.
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- 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.
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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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