influence of co on scenedesmus obliquus chlorella vulgaris and€¦ · oil, pigment content and...

Influence of CO2 on Scenedesmus obliquus, Chlorella vulgaris and

Chlorella protothecoides microalgae growth and evaluation of its

biomass

Joana Marta Leonardo de Assunção

Thesis to obtain the Master Science degree in

Biotechnology

Supervisors: Prof. Dr. Isabel Maria de Sá Correia Leite de Almeida

Dr. Luísa Maria Gouveia da Silva

Examination Committee

Chairperson: Prof. Dr. Leonilde de Fátima Morais Moreira

Supervisor: Dr. Luísa Maria Gouveia da Silva

Members of the committee: Prof. Dr. Helena Maria Rodrigues Vasconcelos Pinheiro

November 2015

iii

Parts of this work were published and presented:

Section 5.1 and 5.2:

Joana Leonardo, Ana Paula Batista, João Manoel, Alberto Reis, Paula Marques, Luísa Gouveia

CARBON DIOXIDE BIOFIXATION AND LIPID ACCUMULATION BY THREE GREEN

MICROALGAE SPECIES AT DIFFERENT CO2 CONCENTRATIONS, was accepted as scientific

poster to participate in Congresso LatinoAmericano de Biotecnología Algal CLABA, ViÑa del Mar

City, Chile (25-29 October/2015)

v

Acknowledgements

I would like to express my special thanks to Doctor Isabel Sá Correia for conceiving me this

great opportunity to work on this microalgae project, as well as Doctor Luísa Gouveia for its expert

advices on this project and encouragement to finished it.. Also to thank all the people that welcomed

me on Laboratório Nacional de Energia e Geologia (LNEG) with open arms- Graça, Natércia, Dr. Ana

Baptista, Dr. Paula Marques, Dr.Paula Passarinho, and all my lab mates. My thanks of gratitude to Dr.

Paula Marques and Doctor Ana Baptista for their help and orientation in the laboratory; and special

thanks to João Manoel for its particular advices, guidance on the lab and to its contribution to the

improvement of my technical skills.

I would also like to thank my family and my friends (the old and the new ones): Pedro Costa,

Margarida Nunes, Bruno Oliveira, Guilherme Benedito, Lucas Ambrosano, Vera Salgado, Catarina

Marques, Catarina Viegas, Ana Morais, Afonso Oliveira, Joana Oliveira, Solange Martins, that are a

crucial part of my life, encouraged me and gave me the motivation to finish this thesis project.

To you all, I am very grateful.

vii

Resumo

O aumento do CO2 na atmosfera tem sido considerado a principal causa do aquecimento

global. As microalgas são microorganismos fotossintéticos que podem mitigar CO2 concomitante com

a produção de biomassa de valor-acrescentado. Neste estudo, Scenedesmus obliquus, Chlorella

vulgaris e Chlorella protothecoides foram cultivadas sob concentrações de CO2 de 0.035%, 5% e 10%

(v/v). Para a Scenedesmus foram também testadas as concentrações 2,5%, 7,5%, 15% (v/v) de CO2.

O objetivo do presente trabalho foi determinar a melhor espécie em termos de crescimento,

biofixação teórica de CO2 (PCO2), teor e perfis de óleos e pigmentos para potencial valorização

económica e aplicação num sistema de biomitigação-CO2. Para a Scenedesmus, foi também

estudada a influência de diferentes velocidades de areação da mistura ar+CO2 (0,25; 0,50; 0,75 e 1

vvm).

A Scenedesmus revelou os melhores resultados em termos de crescimento (µ = 0,99 d-1

)

(7,5% CO2) e produtividade média (0,37 g L-1

d-1

) (2,5% CO2). As diferentes velocidades de areação

de CO2 revelaram resultados de crescimento ambíguos, embora o seu aumento resulte no aumento

da mitigação real. A Scenedesmus mostrou também maior teor em óleo, nomeadamente para o ar

enriquecido com 2,5 e 15% de CO2 (v/v) (26,3%; 25,3% (m/m), respectivamente) e um perfil de ácidos

gordos adequados para a produção de biodiesel, de acordo com a EN 14214.

A C. protothecoides apresentou a maior PCO2 com 1,98 gCO2 L-1

d-1

, obtendo-se grande

concentração de biomassa (5,8 g L-1

) (10% CO2) e foi eficiente na produção de clorofila e luteína.

Palavras chave: microalgas, biomassa, biofixação de CO2, óleos, pigmentos, valorização económica

ix

Abstract

The increasing of CO2 into the atmosphere is considered the main cause of global warming

effect. Since microalgae are photosynthetic microorganisms that can help to CO2 mitigation and at the

same time produce value-added compounds. In this study, Scenedesmus obliquus, Chlorella vulgaris

and Chlorella protothecoides were cultivated under 0.035% (air), 5%, 10% (v/v) of CO2 concentrations.

Scenedesmus was also tested with 2.5%, 7.5%, 15% (v/v) CO2. For Scenedesmus it was also studied

the influence of CO2 aeration rate (0.25, 0.50, 0.75 and 1 vvm). The aim of the present work was to

determine the best microalga in terms of growth kinetic parameters, theoretical CO2 biofixation (PCO2),

oil, pigment content and fatty acid profile for potential economic valorization and application on a CO2-

biomitigation system.

S. obliquus, was the microalga which presented the highest results on growth (µ = 0.99 d-1

),

under 7.5% CO2 and average productivity (0.371 gL-1

d-1

) under 2.5% (v/v) CO2 levels. The study on

different CO2 aeration rates revealed ambiguous results for growth, albeit rising CO2 aerations rates

resulted in real mitigation increasing. Regarding the oil content, Scenedesmus shown the higher

values, namely for the 2.5% and 15% (v/v) CO2 enriched-air with 26.3% and 25.3% (wt.), respectively,

revealing an adequate fatty acid profile for biodiesel production according to EN 14214.

C. protothecoides presented the highest PCO2: 1.98 gCO2L-1

d-1

, producing high biomass

concentration (5.8 gL-1

) (10% (v/v) CO2) and was more targeted to produce chlorophyll and lutein.

Keywords: microalgae, biomass, CO2 biofixation, oils, pigments, economic valorization

xi

Table of contents

Acknowledgements ................................................................................................................................. iv

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

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

Table of contents ..................................................................................................................................... xi

List of Tables ......................................................................................................................................... xiii

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

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

1. General framework .......................................................................................................................... 1

2. Aims ................................................................................................................................................. 2

3. Introduction and literature review .................................................................................................... 3

3.1 CO2 current scenario ............................................................................................................... 3

3.2 Current CO2 capture technologies ........................................................................................... 6

3.2.1 Chemical absorption ........................................................................................................ 6

3.2.2 Solid physical adsorption ................................................................................................. 6

3.2.3 Cryogenic separation ....................................................................................................... 7

3.2.4 Membrane separation ...................................................................................................... 7

3.2.5 Carbon Dioxide Storage/Sequestration (CCS) ................................................................ 7

3.3 Microalgae based carbon mitigation technology ..................................................................... 8

3.3.1 Microalgae biology ........................................................................................................... 8

3.3.2 Biochemical composition ................................................................................................. 9

3.3.3 Why choose microalgae .................................................................................................. 9

3.3.4 Algae strain selection .................................................................................................... 11

3.3.5 Cultivation system.......................................................................................................... 13

3.3.6 Nutritional sources and growth conditions ..................................................................... 15

3.3.7 Microalgae harvesting and downstream processing ..................................................... 20

3.3.8 Applications of the microalgal biomass ......................................................................... 21

4. Materials and methods .................................................................................................................. 23

4.1 Microorganism ....................................................................................................................... 23

4.2 Growth medium composition ................................................................................................. 23

4.3 Inoculum preparation ............................................................................................................. 24

4.4 Microalgae cultivation ............................................................................................................ 24

4.4.1 Photobioreactor assembly ............................................................................................. 24

4.4.2 Cultivation Conditions .................................................................................................... 25

4.5 Determination of growth kinetic parameters and CO2 fixation rate ....................................... 26

4.6 Analytical methods ................................................................................................................ 28

xii

4.6.1 Determination of microalgal biomass growth ................................................................ 28

4.6.2 Lipid extraction and analysis of fatty acid profile ........................................................... 28

4.6.3 Total pigment (chlorophyll and carotenoids) extraction and analysis ............................ 29

5. Results and discussion .................................................................................................................. 32

5.1 Effect of CO2 concentration on microalgae growth ............................................................... 32

5.1.1 OD ................................................................................................................................. 32

5.1.2 Maximum dry cell weight (DCWmax) ............................................................................... 34

5.1.3 Specific grow rate (µ) ..................................................................................................... 35

5.1.4 Average productivity (Pa) ............................................................................................... 36

5.1.5 CO2 biofixation rate ........................................................................................................ 37

5.2 Effect of CO2 concentration on microalgae biomass quality ................................................. 38

5.2.1 Oil content ...................................................................................................................... 38

5.2.2 Fatty acid profile ............................................................................................................ 40

5.2.3 Total pigment content and profile .................................................................................. 44

5.3 Effect of CO2 aeration rates on microalgae growth ............................................................... 46

5.3.1 CO2 biofixation and mitigation ....................................................................................... 48

5.4 Effect of CO2 aeration rates on microalgae biomass quality ................................................. 49

5.4.1 Oil content ...................................................................................................................... 49

5.4.2 Fatty acid profile ............................................................................................................ 50

6. Conclusions and Future work ........................................................................................................ 52

References ............................................................................................................................................ 54

Annex I – CO2 Calibration curve ............................................................................................................ 60

Annex II – Beta-carotene calibration curve ........................................................................................... 61

Annex III – Lutein calibration curve ....................................................................................................... 62

xiii

List of Tables

Table 3.1 - Comparison between different microalgae strains in terms of supplied-CO2% (v/v), temperature

tolerance, biomass productivity (P) (in grams per Liter per day) and CO2 fixation rate (PCO2) (in grams per Liter

per day) (Adapted from Wang et al., 2008). ................................................................................................. 12

Table 3.2 - Comparison between the microalgae open cultivation system and closed photobioreactor designs

(Brennan and Owende, 2010; Chisti, 2007; Schenk et al., 2008). .................................................................. 15

Table 4.1 – Bristol Medium composition. ..................................................................................................... 23

Table 4.2 - Chlorella Medium composition. .................................................................................................. 23

Table 4.3 - Trace solution element composition. .......................................................................................... 24

Table 4.4. CO2 concentrations (v/v) tested in C. vulgaris, C. protothecoides and S. obliquus. ........................... 25

Table 5.1. - Theoretical CO2 biofixation rates between the studied microalgae strains, according to the percentage

(v/v) of CO2 concentration tested. ............................................................................................................... 38

Table 5.2 - Fatty acid profile for the different CO2 concentration (v/v) tested for S. obliquus and respective

standard deviation among the average of two duplicates. ............................................................................. 41

Table 5.3 - Fatty acid profile for the different CO2 concentration (v/v) tested for C. vulgaris, with respective

standard deviation among the average of two duplicates. ............................................................................. 42

Table 5.4. - Fatty acid profile for the different CO2 concentration (v/v) tested for C. protothecoides with the

respective standard deviation among the average of two duplicates. ............................................................. 43

Table 5.5 - Fatty acid profile for the different aeration rates tested for S. obliquus [5% (v/v) CO2 concentration; ≈

28ºC; 74 µmol photons m-2

s-1

] and respective standard deviation among the average of two duplicates. .......... 50

xv

List of Figures

Figure 3.1 - Representation of the sharp increase of CO2 levels in parts per million (ppm), in the atmosphere,

since the late 50’s till 2015. Source: NOAA/ESRL, 2015. ........................................................................................ 4

Figure 3.2 - (A) CO2 emissions per country from fossil fuel use and cement production (B) Projections on the

primary energy consumption by regions in billion toe (tone of oil equivalent). Adapted from Energy Outlook 2035,

BP (2015). ............................................................................................................................................................... 4

Figure 3.3 - World CO2 emissions by sector (in billion tones CO2) and respective projections till 2035 (Source:

Energy Outlook 2035, BP (2015)). ........................................................................................................................... 5

Figure 3.4 - Schematic description of photosynthetic conversion of CO2, solar energy and wastewater, into a

variety of valuable end products, by microalgae (Ho et al., 2011a). ...................................................................... 11

Figure 3.5 – Different microalgae cultivation systems (A) raceway pond (B) tubular PBR (horizontal) (C) tubular

PBR (inclined) (D) column PBR (E) flat-plate PBR. ............................................................................................... 14

Figure 4.1 - (A) Bubble column PBR assembly (1 – Inlet air flow; 2 – Output to collect samples; 3 – Output air

flow; 4 – Inoculum flask); (B) PBRs during microalgae cultivation experiments..................................................... 25

Figure 4.2 - Schematic diagram of the experimental setup. .................................................................................. 26

Figure 4.3 - (A) Sample to centrifuge; (B) Sample after centrifugation; (C) Biomass collected to the Petri dish. ... 28

Figure 5.1 - Growth curves of the cultures of Scenedesmus obliquus, Chlorella vulgaris and Chlorella

protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

; 1vvm) subjected to different CO2 concentrations tested: [Air (

); 2.5% CO2 ( ); 5% CO2 ( ) 7.5% CO2 ( ); 10% CO2 ( ); 15% CO2 ( )]. Vertical bars represent

standard deviations among the average of two duplicates. ................................................................................... 33

Figure 5.2 – Representation of the maximum dry cell weight (g L-1

) of the cultures of S. obliquus, C. vulgaris and

C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

;1 vvm) subjected to different CO2 concentrations tested: Air

( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ).

Vertical bars represent standard deviations among the average of two duplicates. .............................................. 34

Figure 5.3 - Representation of the specific growth rate (d-1

) of the cultures of S. obliquus, C. vulgaris and C.


s-1

; 1vvm), subjected to different CO2 concentrations tested: Air ( );

2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical

bars represent standard deviations among the average of two duplicates. ........................................................... 35

Figure 5.4 - Representation of the average productivity (g L-1

d-1

) of the cultures of S. obliquus, C. vulgaris and C.


s-1

; 1vvm), subjected to different CO2 concentrations tested: Air ( );

xvi

2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical

bars represent standard deviations among the average of two duplicates. ........................................................... 36

Figure 5.5 - Oil content in % (mass of oil per mass of dried biomass) from the cultures of S. obliquus, C. vulgaris

and C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

, 1vvm), subjected to the different CO2 concentrations

tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2

( ). Vertical bars represent standard deviations among the average of two duplicates. ...................................... 39

Figure 5.6 - Fatty acid profile from the oil of one of the duplicates of C. protothecoides [5% (v/v) CO2

concentration, ≈ 28º C; 74 µmol photons m-2

s-1

; 1vvm], obtained by GC. ............................................................ 40

Figure 5.7 – Representation on the total chlorophyll content (mg L-1

) on the extracts, from the cultures of S.

obliquus, C. vulgaris and C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

; 1vvm), subjected to different CO2

concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2

( ); 15% (v/v) CO2 ( ). Vertical bars represent standard deviations among the average of two duplicates. ...... 44

Figure 5.8 – Representation of the content (mg L-1

) on beta-carotene (A) and lutein (B) pigments, on the extracts

from the cultures of S. obliquus, C. vulgaris and C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

; 1vvm),

subjected to different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v)

CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical bars represent standard deviations among the

average of two duplicates. ..................................................................................................................................... 45

Figure 5.9 – Representation of the biomass performance of S. obliquus [5% (v/v) CO2 ; ≈ 28º C; 74 µmol photons

m-2

s-1

], under different CO2 flow rates: 0.25 vvm ( ); 0.50 vvm ( ); 0.75 vvm ( ); 1 vvm ( ) in terms of (A)

Maximum dry cell weight; (B) Specific growth rate; (C) Average productivity. Vertical bars represent standard

deviations among the average of two duplicates. .................................................................................................. 47

Figure 5.10 – Comparison between the theoretical CO2 biofixation ( ) and the real CO2 biofixation ( ), along the

different aeration rates tested, in S. obliquus [5% (v/v) CO2 concentration; ≈ 28ºC; 74 µmol photons m-2

s-1

].

Vertical bars represent standard deviations among the average of two duplicates. .............................................. 49

Figure 5.11 - Oil content in % (mass of oil per mass of dried biomass) from the cultures of S. obliquus [5% CO2

(v/v) concentration; ≈ 28ºC; 74 µmol photons m-2

s-1

], subjected to different CO2 flow rates: 0.25 vvm ( ); 0.50

vvm ( ); 0.75 vvm ( ); 1 vvm ( ). Vertical bars represent standard deviations among the average of two

duplicates. ............................................................................................................................................................. 50

xvii

Abbreviations

CCMs Carbon concentration mechanisms

CCS Carbon Capture Storage

CO2 Carbon dioxide

GHE Greenhouse effect

GHG Greenhouse gases

GtCO2 Billion tones of CO2

DCWmax Maximum dry cell weight

DIC Dissolved inorganic carbon

EC Electrocoagulation

EU European Union

MEA Monoethanolamine

MWh Megawatt-hour

NOAA National Oceanic and Atmospheric Administration

NH3 Ammonia

NOx Nitrogen oxides

OD Optical density

Pa Average biomass productivity

PCO2 Maximal theoretical CO2 fixation rate

Pmax Maximum biomass productivity

PBR Photobioreactor

Ppm Parts per million

RCO2 Real CO2 biofixation

SOX Sulphur oxides

Toe Tonne of oil equivalent

Vvm Volume of gas per volume of culture per minute

WMO World Meteorological Organization

µ Specific growth rate

1

1. General framework

Greenhouse gases (GHG) are accumulating dramatically in the atmosphere as a result of

excessive use of fossil fuels in human activities and industrialization. Carbon dioxide (CO2) is the

major responsible for the greenhouse effect (GHE). The increasing concentration of CO2 in the

atmosphere is considered to be among the greatest problem in regards to global warming and

consequently, climate change. In this context it is important to find viable and sustainable solutions to

mitigate or neutralize CO2.

There are several available technologies to remove/manage the CO2 emitted to the

atmosphere. These include chemical adsorption, solid physical absorption, cryogenic and membrane

separation, and carbon dioxide storage (direct injection of CO2 into the deep ocean, geological

formations, etc). These methods still have disadvantages, since they are not technical nor

economically feasible. Hence, the biological mitigation emerged as a potential economically feasible

and environmentally sustainable technology to mitigate CO2.

Microalgae have been reported as a valuable biotechnological resource to use CO2 as a

nutrient to growth autotrophically. Microalgae are aquatic organisms capable of using light, water and

nutrients, to perform photosynthesis and produce organic biomass. They share with plants the same

pigments responsible for photosynthesis, however do not present organs, stems, leafs, fruits that are

energetically expensive to produce. Compared to terrestrial plants, they are capable to grow faster

and have higher CO2 biofixation rates, do not need arable land, neither compete with crops, nor

potable water and have the interesting feature to produce biological derivatives (e.g., oils, sugars,

proteins, pigments) with substantial value application in the widest range of industrial sectors

(food/feed, biofuels, cosmetic, health, energy, etc).

For these reasons, microalgae is an appealing resource to, ultimately, mitigate CO2 from flue

gases, taking full advantage from the produced biomass and respective by-products, to economic

valorization, allowing the creation of a local bio-economy, intrinsically bound to a Biorefinery concept

and the promotion of green quotas in the future.

2

2. Aims

The aim of the present work is to determine among the microalgae species tested, the best

one, in terms of growth (growth kinetic parameters), average biomass productivity, theoretical CO2

biofixation, oil content and fatty acid profile to be used into biodiesel production and pigment

accumulation (for economic valorization). Three different species of microalgae: Scenedesmus

obliquus, Chlorella protothecoides and Chlorella vulgaris were tested under different CO2

concentrations: air [0.035% (v/v)], 5% (v/v), 10% (v/v) at flow rate of 1 vvm (volume of gas per volume

of culture per minute). The growth efficiency (maximum dry cell weight, specific growth rate, biomass

average productivity), the theoretical CO2 biofixation, the oil and fatty acid profile and pigment content,

total chlorophylls, pigment profile and quantification, were assessed.

Due to a better performance of Scenedesmus obliquus, this microalga was selected to

perform tests with different CO2 aeration rates (0.25, 0.50, 0.75 and 1.0 vvm) at 5% (v/v) of CO2

enriched-air, to verify the real CO2 mitigation and the influence on grow productivities, lipid content and

fatty acid profile. Other different CO2 concentrations were tested on this microalgae (2.5, 7.5% and

15% (v/v) of CO2) to evaluate the parameters described previously.

This study aims to further tests on a pilot scale (in a bio-mitigation system), using flue gas

effluents for CO2 retention/bioremediation. Consequently, it intended to take full advantage of the

products that could be obtained in those conditions, affording environment benefits and contributing to

the bioeconomy.

3

3. Introduction and literature review

3.1 CO2 current scenario

During the past few years, global energetic future has become an important topic of debate.

Nowadays, all global industrialized societies are based on non-renewable resources,

accounting for 80% of primary energy consumption. The intensification and utilization of fossil fuels

(coal, oil and natural gas) for industry, transport and electricity/power generation have been increasing

every year that goes by, driven by an increased energetic demand, due to growth on industrialization

and global population. BP estimates that world population will reach 8.4 billion by 2035, meaning that

more than 1.6 billion people will need energy. This predicts an increasing on the global energy

consumption by 37% from 2013 to 2035, what could lead to the reserve depletion and trigger an

energetic crisis (BP, 2015).

The intensive utilization of fossil fuels is reflecting the dramatic accumulation of GHG

(Greenhouse Gases) into the atmosphere. CO2 has become the major focus of attention because of

its position as the primary GHG. This gas is naturally present in the atmosphere, as part of the Earth's

carbon cycle (its natural and complex interaction among the atmosphere, soil, oceans, plants and

animals). However, since pre-industrial times, due to human activities (fossil fuel use and land use

changes – global deforestation and loss of arable land), this cycle start to become affected both by

adding more CO2 to the atmosphere and by influencing the ability of natural sinks (e.g. forests and

oceans) to remove CO2 from the air (Dhillon and von Wuehlisch, 2013). The most obvious and

immediate effect is the so called global warming and consequently, the negative implications on

climate change (Chiu et al., 2009). Global surface temperatures have increased by about 0.8 ºC since

1880 (IPPC, 2001). This impressive alteration has having serious consequences on level sea rising,

melting of ice caps, ocean acidification and extreme climate events with greater incidence and

magnitude, such as precipitation (floods), storms, drought, heat waves, hurricanes and tornados

(IPPC, 2007). Evidences of ecosystems perturbations have been dictating changes on animal

migration patterns (Parmesan, 2006) combined with an alarming biodiversity decline with

unprecedented extinction rates (Ceballos et al., 2015). As consequence, the way where species live

and how they interact, can suffer a profound transformation on current ecosystems, posing great risk

on Planet sustainability.

According to WMO (World Meteorological Organization), the CO2 accumulation has reached

the highest rate since 1984. Between 2012 and 2013, the increase was 2.9 ppm, compared to an

average of 1.5 ppm per year, since 1990, and 2.1 ppm per year in the last decade (WMO, 2014).

Recent data from NOAA (National Oceanic and Atmospheric Administration) (Figure 3.1) revealed

that in 2015, for the first time, CO2 concentrations surpassed the value of 400 ppm (NOAA/ESRL,

2015). This threshold is of symbolic significance, since forecasts point out that CO2 levels greater than

4

450 ppm would be ‘dangerous’ and could be highly destructive to the world’s global climate (Hansen

et al., 2007).

Regarding the main responsible for the CO2 emissions to the atmosphere, just only in 2013,

China (10.3 billion tonnes CO2 or 29%), United States (5.3 billion tonnes CO2 or 15%) and European

Union (EU28) (3.7 billion tonnes CO2 or 11%) account for more than half (55%) of the total CO2

emissions (Figure 3.2A). Also, due to strong development (increment on industrialization and

population) and continuous energy needs, the emerging economies (e.g. China, India) had been

recorded an accentuated increase on fossil energy consumption, reflecting the global growth of CO2

emissions during the last decades (BP, 2013). According to BP (Figure 3.2B), China and India will

continue to be the key drivers of non-OCDE growth and are projected to grow by 5.5% per annum

between 2013 and 2035 (BP, 2015).

Figure 3.1 - Representation of the sharp increase of CO2 levels in parts per million

(ppm), in the atmosphere, since the late 50’s till 2015. Source: NOAA/ESRL, 2015.

Figure 3.2 - (A) CO2 emissions per country from fossil fuel use and cement production

(B) Projections on the primary energy consumption by regions in billion toe (tone of oil

equivalent). Adapted from Energy Outlook 2035, BP (2015).

5

Currently, the power generation and the industrial sectors are the major responsible for almost

two-thirds of the global CO2 emissions (Figure 3.3). Since they have to fulfill their large power

requirements, industries related to electricity generation, natural gas processing, cement, iron and

steel manufacturing, combustion of municipal solid waste have a great slice as major contributors of

atmospheric CO2 emissions. Projections till 2035 point out an increasing trend if climate policies were

not taken in time (BP, 2015).

Figure 3.3 - World CO2 emissions by sector (in billion tones CO2) and respective projections till 2035 (Source:

Energy Outlook 2035, BP (2015)).

In this context, the Governmental Institutions have been defining priority plans for the

implementation of energetic measures, based on neutral or reduced carbon emissions. These

measures, include a more rational management of available resources and a strong investment on

several renewable energies – solar, geothermal, wind, hydroelectric, ocean wave and biomass

(bioenergy/biofuels) - that are being developed as more sustainable alternative energy sources when

compared with the fossil fuel combustion (Sayre, 2010). This is the approach that is intended to

improve the current energetic world panorama. Concerning climate change, renewable energy and

follow-up the Kyoto’s Protocol goals (which ended in 2012), the European Union (EU), created the

Directive 2009/28/CE. It establishes that, till 2020, each European Member-State has to reduce 20%

of the greenhouse gases emissions (against the reference-year 1990); has a mandatory quota of

20% of energy from renewable sources in the internal electricity market and has to improve energetic

efficiency in 20%, the so called EU “20-20-20” targets. Another set target, concerns the transport

sector: each Member-State is expected to produce, at least, 10% of fuels from renewable energy

sources (biofuels), incorporating them into the gasoline and diesel, till 2020.

The global awareness of those reported issues, such as the reduction of fossil fuel

consumption, lowering the GHG emissions and the urgency to manage the climate risk, have been

increasing the research and development of several long-term air treatment technologies to neutralize

or mitigate CO2 from the atmosphere.

6

Since power generation and industrial sectors are sources with large emission volumes, this

make them amenable to the addition of CO2 capture technologies, becoming possible to make a local

treatment as part of the global strategy to mitigate/neutralize the GHG.

3.2 Current CO2 capture technologies

The capture of CO2 from air has been studied for decades in the context of producing CO2-

free air (Falkowski, 1994). The extraction of CO2 from air is possible, but not practical, due to high

degree of dilution, (approximately 400 ppm by volume). This requires a more developed and efficient

technology (Rahaman et al., 2011). Capturing CO2 from pollutant industries e.g. cement, power

stations, incinerators, biomass energy facilities, synthetic fuel plants, natural gas processing, and

fossil fuel-based hydrogen production plants is probably the most effective (IPPC, 2005). However,

based on current technology, there is no single way to capture CO2 in a sustainable manner (Brennan

and Owende, 2010). The available technologies either consume huge amount of energy, are no cost

effective when up-scaled to a commercial stage or have disposal problems (Rahaman et al., 2011).

3.2.1 Chemical absorption

In industry, chemical absorption is the most common used process to separate CO2 from a

flue gas. This method is based on the dissolution or solubility of gas into liquid phase and a

monoethanolamine (MEA) solution is been used (Pires, et al., 2011). When the solvent reaches its

maximum absorption capacity, it is applied a heat treatment between 100-150ºC, to release CO2 for

storage and regenerate the MEA solution. However this process has a great disadvantage due to high

energy consumption during MEA desorption which represents up to 70% of the total operating costs in

CO2 capture plant. Besides, MEA can easily react with SO2 from the flue gas, causing irreversible

degeneration of MEA and originating corrosive products, which can damage the equipment when in

contact with O2 (Lam et al., 2012).

3.2.2 Solid physical adsorption

This process utilizes carbonaceous adsorbents like activated carbon, X-type zeolite, CaO, etc.

X-type zeolite, for instance, has revealed to be an excellent choice in the adsorption of both low and

high CO2 concentrations (Chatti et al., 2009). Nevertheless, the main disadvantage of using solid

adsorbents is the need to pre-treat the flue gas. This requirement is fundamental, because most of

flue gases have high moisture content, together with contaminants (e.g. SOx and NOx), which can

interfere with the subsequent adsorbent regeneration. Moreover, the stronger the affinity of the

adsorbent, the harder it becomes to desorb the gas, resulting in an increased heat energy requirement

for recycling and reusing the adsorbent in the next cycle (Mérel et al., 2006).

7

3.2.3 Cryogenic separation

Cryogenic separation is one of the most preferred technologies to produce pure gases due its

high efficiency. This technology allows the capture of CO2 at a yield higher than 95% (Hart and

Gnanendran, 2009) and a gas purity of 99.99% (Paranjape et al., 1998). The cooling and

condensation/distillation at certain temperature and pressure of the multigaseous components is the

base of this technique. The main drawback is the high energy requirement to provide enough

refrigeration to the process. In addition, moisture has to be removed before flue gas is channeled to

the cooling unit, to avoid plugging by ice or an excessive rise in pressure drop during operation (Lam

et al., 2012).

3.2.4 Membrane separation

The membrane acts as a filter, separating desired gases from a mixture of gas stream based

on membrane’s permeability and selectivity. Membrane based-processes for capturing CO2 can offer

several practical advantages, including high selectivity at low cost operations. At the moment, the

mostly used membrane materials are polymers. Polymeric membranes have been reported as

consuming less energy during the separation process compared to absorption processes. On the

other hand, performance on this kind of membrane can be affected by operating temperature. It

means that cooling the flue gas is crucial to maintain the optimum separation process efficiency which

is energy-intensive. Other limitations include high membrane manufacturing cost and fouling effect

(Pires et al., 2011).

3.2.5 Carbon Dioxide Storage/Sequestration (CCS)

Carbon Capture and Storage or Carbon Capture and Sequestration (CCS) is a technology

consisting on the direct capture of CO2 from industrial and energy-related sources, transport to a

storage location and long-term isolation from the atmosphere. The CO2 would then be compressed

and injected into geological formations (oil and gas reservoirs, coal mines, saline aquifers, etc), in the

deep ocean or in mineral carbonates as limestone (IPPC, 2005). However, safety issues remain the

major concern of this technology. CO2 leakage, either rapid or slow, will definitely bring drastic impacts

towards humans and the environment. In addition, continuous monitoring of the entire CO2 storage

reservoirs adds a significant cost to this technology (Lam et al., 2012).

Other approach to carbon sequestration/mitigation is to increase the carbon uptake stored in

terrestrial ecosystems with renewable biomass (Pacala and Socolow, 2004). Forests, energetic crops,

farmlands, croplands, etc. can be used as sink of CO2. Renewable biomass could be a potential

solution to produce environment friendly and economical biofuels, or even used as raw material to

produce heat/electricity, helping to neutralize the carbon emissions. Nevertheless, this strategy has

not global feasibility, as it only retains one small fraction of CO2 and will not keep up with the global

energy demand. Competition between crop areas required for energy and food purposes could trigger

an increase of price of raw materials and if it is taken to the extreme, deforestation, loss of biodiversity

8

and natural habitats could be aggravated, and turn out to be even worse in terms of climate

interference.

3.3 Microalgae based carbon mitigation technology

Biological mitigation of atmospheric CO2 has received attention in recent years. Biofixation of

CO2 can be performed either by plants or photosynthetic microorganisms, such as bacteria or

microalgae (Chiu et al., 2009; Dragone et al., 2011). Microalgae have been reported as a potential

biotechnological resource to mitigate/bioremediate CO2.

3.3.1 Microalgae biology

Microalgae are a large and diverse group of simple aquatic and photosynthetic

microorganisms. They are primitive plants (tallophytes) characterized by lacking roots, stems and

leaves and have chlorophyll a as their primary photosynthetic pigment. They are ubiquitously

distributed throughout the biosphere and grow under the widest possible variety of conditions from

freshwater to extreme salinity (Kumar et al., 2011). Their morphology ranges from unicellular,

filamentous, colonial and multicelullar. Algae can be autotrophic, heterotrophic or mixotrophic.

Autotrophic algae use photosynthesis to harness sunlight and fix the inorganic carbon from

atmospheric CO2 which is then assimilated in the form of reserve materials such as carbohydrate,

lipids and proteins. Some species are heterotrophic requiring an external source of organic

compounds as nutrients to turn them into their building blocks. Certain algal species have the ability to

perform both photosynthesis and acquire exogenous organic nutrients, a process called mixotrophy

(John et al., 2011).

Microalgae are mainly phototrofic, playing an important role in nature as primary producers.

Based on photosynthesis mechanism they convert water, carbon dioxide, light and other few minerals

such as N, P, K, Mg, and S into oxygen and biomass (Madigan et al., 2010).

Most of these microorganisms are cyanobacteria (prokaryotic cells) or eukaryotic cells that

comprise many different type of classes categorized by their pigmentation, life cycle and basic

structure (Khan et al., 2009). The most important classes are green algae (Clorophyta), red algae

(Rhodophyta) and diatoms (Bacillariophyta) (Brennan and Owende, 2010).

Green algae are more closely related to the plants, they have the same pigments (chlorophyll

a and b and carotenoids), a well defined nucleus, the same chemicals in their cell walls (cellulose),

and the same storage product (starch) (Madigan et al., 2010).

In a multistep process of photosynthesis, plants and microalgae fix CO2 into sugar using light

and water as energy and electron source, respectively. The overall reaction for photosynthesis is

given by the equation below:

9

3.3.2 Biochemical composition

In general, biochemical composition of microalgae comprises the following primary

components: proteins, carbohydrates (starch, sugars, glucose, etc) and lipids (typically composed by

glycerol, sugars or bases esterified to fatty acids having carbon numbers in the range of C12–C22).

Also, encompass chlorophylls as primary pigments and secondary metabolites, such as pigment

astaxanthin, beta-carotene, lutein, etc. and vitamins. Microalgae seem to have the ability to undergo

programmatic in photosynthetic carbon partitioning mechanism in response to changes on the

environmental conditions. Thus biochemical composition, particularly in the relative amounts of crude

protein, lipids and carbohydrate per total organic matter basis, fluctuates in a wide range of

proportions, deeply depending on the specie and also on the culture conditions (Dean et al., 2010).

3.3.3 Why choose microalgae

Compared to other plant feedstock, microalgae have a number of advantages in CO2

mitigation and bio-oil generation. These include high photosynthetic conversion efficiencies; rapid

biomass production; ability to thrive in diverse ecosystems, even under extreme edafo-climatic

conditions (Chisti, 2007a); capacity to produce a wide variety of biofuels; non-competition for

agricultural areas (marginal lands such as deserts, rocky areas and salt pans can be used) nor with

crops, non-competition for drinking waters (saltwater, brackish water and wastewaters can be used);

harvesting routines can be carried out daily with a better equipment and resources management

trimming storage costs and a notable environmental bioremediation potential, such as CO2 biofixation

from the atmosphere and flue gas and/or wastewater treatment.

Microalgae have a much higher growth rate (100 times greater) than the most land-based

plant due to their higher photosynthesis efficiency. They reproduce rapidly multiplying within 24 hours

(Rahaman et al., 2011) and they have around 10-50 times higher CO2 biofixation rates than terrestrial

plants, primarily due to more chlorophyll per unit area (Raeesossadati et al., 2014). Unlike unicellular

microalgae, plants have supportive structures such as stems, roots or fruits that are energetically

expensive to growth and to produce biofuels. Additionally, microalgae have carbon concentrating

mechanisms (CCMs) that, in some species, suppress photorespiration (Jansson and Northen, 2010;

Spalding, 2008) and their metabolic flexibility offers the possibility to modify their biochemical

pathways (e.g. towards protein, carbohydrate or oil synthesis) and cellular composition (Tredici, 2010).

Microalgae cultivation has been investigated as an additional step in flue gas treatment,

aiming for reduction of CO2 levels in the exhaust flue gas. Previous studies have shown that

microalgae can be successfully employed for the treatment of simulated flue gases or flue gases

emitted from coal-fired power plants, municipal waste incinerators, gas boilers or industrial heaters

using kerosene as fuel (Anjos et al., 2013). The direct injection of flue gasses into ponds increases

10

biomass productivity by 30% compared with direct injection of an equivalent concentration of pure CO2

(Douskova et al., 2009).

The efficiency of CO2 capture by microalgae can vary according to the state of the algal

physiology, reactor design and temperature. Carbon-dioxide capture efficiencies as high as 80% to

99% are achievable under optimal conditions and with gas residence times as short as two seconds

(Keffer and Kleinheinz, 2002). For a typical 200 Megawatt-hour (MWh) natural gas-fired power plant, it

has been estimated that an algal pond of 3600 acres would be sufficient to capture 80% of the plant's

CO2 emissions during daylight hours, assuming an algal biomass productivity rate of 20 gm-2

d-1

(Herzog and Golomb, 2006). Because of the greater CO2 emission levels per MWh of coal-burning

power plants, a pond approximately 7000 acres in size should be required to capture 80% of the CO2

emissions from a 200-MWh coal-burning power plant during the day (Sayre, 2010).

Locating photobioreactors (PBRs) near CO2 point sources can provide several potential cost-

and energy-saving advantages. Integrated power plant-algal reactor facilities would reduce the costs

of CO2 transportation, produce limited waste heat from the power plant for warming the PBRs in the

winter, and could give carbon credits to the utility. Locating PBRs near CO2 sources can be

problematic, however, depending on land availability and the climatic suitability of the site. Thus, the

utilization of microalgal species with high ability to biofixate CO2 could reduce significantly the flue gas

treatment costs (Sayre, 2010).

Biofuels encompass a great alternative to reduce the global carbon footprint. Production of

biofuels from microalgae is a great alternative to the so called first and second generation biofuels.

First generation biofuels are produced from agricultural feedstocks that can also been used as food

and feed purposes. For example, bioethanol is one of the most common liquid biofuels currently being

produced mainly from food crops (rapeseed, sugarcane) raising the issues of food competition, arable

land usage and freshwater supply. Second generation biofuels are produced from lignocelullosic

biomass, woody crops, agricultural residues or waste, although this approach has a major drawbacks

because the technology to convert lignocelluloses to liquid biofuels efficiently is still lacking and

therefore it is not yet economically viable (John et al., 2011). As opposed to land-based biofuels,

microalgae are considered as third generation biofuels meaning that does not imply the use of

agriculture lands and requiring a relatively small amounts of water (Brennan and Owende, 2010).

Some microalgae species, under suitable culture conditions, are able to accumulate up to 50-70% of

oil per dry weight (Chisti, 2007b), increasing their heat of combustion and fuel value. Algae-based

biofuels have an enormous market potential that may contribute to diminish fossil fuel importations,

hence reducing country’s dependence (Sjors van Iersel et al., 2010).

Beyond biofuel-based microalgae, these microorganisms are also other important added by-

product producers. Collected microalgal biomass can be turned into different by-products such as

vitamins, pigments, proteins, sugars, etc. and can be processed by agricultural, food, chemical,

cosmetic and energy industries into fertilizer, food, nutraceuticals, feed, cosmetics, respectively,

among others. This could be achieved with sustainable inputs, with local production and lower impact

on natural resources (Sjors van Iersel et al., 2010). Moreover, selling these products could give an

11

extra profit to support this technology within the Biorefinery concept and could contribute to attract

investments increasing the bioeconomy of this CO2 mitigation technology (Sen, 2012).

The microalgae CO2 bio-mitigation could be made more economically cost-effective and

environmentally sustainable, if concomitant with wastewater treatment. Wastewaters can be used to

cultivate microalgae, with a series of advantages: they can provide a pathway for removing nitrogen,

phosphorus and metal from wastewater, since several studies have been shown their efficacy to

remove such compounds; it can lead to a saving in terms of chemicals utilization such as nitrate and

phosphate as exogenous nutrients (which have high energy-intensive production) and ultimately, it

can result in precious freshwater resources savings (Wang et al., 2008). The co-digestion of

microalgae with wastewater sludge for biogas production should also be considered, because this

strategy could be integrated into the existing wastewater infrastructure (Znad et al., 2005).

The combination of CO2 biofixation and wastewater treatment concomitant with high-value

compounds and biofuel production may thus provide a very promising alternative to current CO2

mitigation strategies as it shown on Figure 3.4.

3.3.4 Algae strain selection

The selection of culture strains are the foremost important aspect to take care for the

mitigation of CO2. Microalgae are a biological population that contains a variety of strains. The

expectations indicated to nearly more than 50,000 microalgae species in the Earth ecosystems

(Brennan and Owende, 2010), but just only around 100 have been studied and analyzed, and around

5 are commercialized (Olaizola, 2003). However not all species are suitable for the carbon mitigation

process. Important criteria such as high growth rates, temperature range, resistance to shear stress,

ease of collect during harvesting process, preferentially associated with spontaneous settling or bio-

flocculation characteristics and do not posing potential risk, high potential to produce useful by-

products, decide the aptness of strain for CO2 mitigation.

Flue gases from industry have between 10% to 20% of CO2 concentration (Tick, 2010).

Therefore it would be crucial to choose microalgae that are tolerant to relatively high CO2 levels for

biofixation (Maeda et al., 1995). Dunaliella tertiolecta, Chlorella protothecoides, Chlorella vulgaris,

Figure 3.4 - Schematic description of photosynthetic conversion of CO2, solar

energy and wastewater, into a variety of valuable end products, by microalgae

(Ho et al., 2011a).

12

Scenedesmus obliquus and Scenedesmus sp. (all belonging to the Chlorophyceae class) have been

identified as promising microalgae strains capable of assimilate significant quantity of CO2. The listed

microalgae strains are well known for their adaptability towards surrounding environment and able to

endure extreme growing conditions (Lam et al., 2012).

Table 3.1 shows a comparison between different microalgae strains used for mitigation of

CO2, in terms of biomass productivity and CO2 fixation rate, both in g L-1

per day, applied at different

conditions of CO2 percentages (v/v) and temperatures. Some microalgae can tolerate up to 40% CO2-

enriched air (v/v). Chlorella sp. seem to be a good candidate to apply in a CO2 mitigation system,

since reveal a good performance in terms of biomass productivity and presents high CO2 biofixation

rates, above 1g L -1

per day. Comparing Botryococcus braunii and Scenedesmus sp. under flue gas

conditions, it was found that Scenedesmus sp. is the most suitable for CO2 mitigation due to high rates

of biomass production (0.203 g L-1

d-1

). B. braunii and Scenedesmus sp. were found to grow better

using flue gas when compared to air enhanced with CO2 (Yoo et al., 2010).

The selection of suitable microalgae strain for CO2 mitigation has significant effect on efficacy

and cost competitiveness on the bio-mitigation process.

Table 3.1 - Comparison between different microalgae strains in terms of supplied-CO2% (v/v), temperature

tolerance, biomass productivity (P) (in grams per Liter per day) and CO2 fixation rate (PCO2) (in grams per Liter

per day) (Adapted from Wang et al., 2008).

Microalgae CO2 %

(v/v)

Temperature (ºC)

P (g L

-1d

-1)

PCO2 (g L

-1d

-1)

References Specific remark

Chlorococum littorale 40 30 - >1.0 Iwasaki et al., 1998; Murakami and Ikenouchi, 1997

C. kessleri 18 30 0.087 - de Morais and Costa, 2007b

Chlorella sp. UK001 15 35 - > 1.0 Murakami and Ikenouchi, 1997

C.vulgaris 15 27 - 0.62 Yun et al., 1997 Artificial wastewater

C. vulgaris Air 25 0.040 - Scragg et al., 2002 Watanabe’s medium

C.vulgaris Air 25 0.024 - Scragg et al., 2002 Low-N medium Chlorella sp. 40 42 - 1.0 Sakai et al., 1995 Chlorella sp. Air 26 0.682

a - Chiu et al., 2008

Chlorella sp. 2 26 1.445a - Chiu et al., 2008




Chlorella sp. - - - 1.38 Zhao et al., 2011 Bubble photobioreactor

Dunaliella sp. 3 27 0.170 - Kishimoto et al., 1994 High salinity, carotene

D. tertiolecta 10 - 0.270 - Sydney et al., 2010 Fermentor

H. pluvialis 16-34 20 0.076 0.14 Huntley and Redalje, 2007

Commercial scale, outdoor

S. obliquus Air - 0.009 0.02 Voltolina et al., 2005 Wastewater, outdoor, winter

S.obliquus Air - 0.016 0.03 Voltolina et al., 2005 Wastewater, outdoor, winter

Scenedesmus sp. 25 Flue gas 0.203 - Yoo et al., 2010

13

a Culture incubated for 4-8 days

3.3.5 Cultivation system

Various growth systems have been used in order to isolate and cultivate microalgae in the

laboratory, as well as outdoors, on small or large scale production. For the cultivation of microalgae for

CO2 mitigation both open and closed systems are used. Open systems can be categorized into natural

waters (lakes, lagoons) and artificial ponds or containers. Raceways are the most commonly used as

artificial system (Cossı et al., 2003). This is a shallow system (10-50 cm), allowing the penetration of

atmospheric CO2 and natural light. In most designs some form of mixing and circulation equipment is

required to stabilize microalgae growth and prevent sedimentation. They are relatively cheaper to build

and easy to operate. However this type of system presents a wide number of disadvantages, including

the requirement for large cultivation areas, difficulty on controlling cultivation conditions (light

availability, temperature, water evaporation, etc.) (Costa et al., 2006) and has low CO2 diffusion rates

from the atmosphere. In addition it can potentiate contaminations, like unnecessary algae or protozoa,

reducing the biomass productivity and making this system inadequate for the production of some

commercial refined products (Ugwu et al., 2005). In order to overcome some of these difficulties,

ponds could be covered with a transparent material (Jorquera et al, 2010). This covering could

facilitate the contention of CO2 in the medium, trapping CO2 losses, and avoid contaminations,

resulting in an improved microalgal populations and biomass productivity (Richmond et al., 1993).

However an important issue is addressed when it refers to minimization of CO2 losses. CO2

concentration in the culture medium should not drop below the critical CO2 value required for

microalgal growth at any point, because when it becomes too limiting, photosynthesis is restricted

(Suh and Lee, 2003).

Due to these drawbacks, an alternative is the use of closed PBRs. Examples of closed PBRs

are airlift, bubble column, tubular reactor, flat-plate etc. and generally agitation is done non-

mechanically. These PBRs tend to be more complex and expensive than open systems, but allow for

better control of the algae culture environment. They can reduce to close to nule contamination (work

at nearly sterile conditions), can be successfully operated to cultivate single species, allows a high

biomass concentration, can be constructed over any open space, offer better control of the

temperature and pH and reduce CO2 and/or water losses (Chisti, 2007; Raven, 2003). The proper

penetration of CO2 into the PBR enables the microalgae to easily consume the carbon atoms and

growth regularly. Also, bioreactors suitable for CO2 mitigation have the flexibility of using CO2 rich gas

as a means of mixing as well as providing nutrient for the microalgae growth.

B. braunii 25 Flue gas 0.077 - Yoo et al., 2010 B. braunii - 25-30 1.100 >1.0 Mukarami and

Ikenouchi, 1997 Accumulating hydricarbon

B. braunii 10 - 0.50 Sydney et al., 2010 Fermentor S.obliquus 18 30 0.140 0.26 de Morais and Costa,

2007a

14

Different geometric/design features of PBR define different performances of CO2 mitigation

process and consequently, biomass production (Kunjapur and Eldridge, 2010).

PBRs consists of an array of transparent tubes (either plastic or glass) (Ugwu et al., 2008),

allowing the penetration of the light in the dense medium. The tubular array can be aligned horizontally

(Molina et al., 2001), inclined (Sa et al., 1999), as helix (Watanabe and Saiki, 1997) or vertically

(commonly known as column photobioreactor) (Tanaka, C. U., Ugwu, J. C., Ogbonna, 2002) and

tubes are generally 0.1m or less in diameter (Chisti, 2007b). Cultures of microalgae are re-circulated

either with a mechanical or air-lift system, the latter allowing CO2 and O2 exchanges between the liquid

medium and aeration gas as well as providing a mechanism for mixing (Eriksen, 2008).

The flat-plate systems are other type of PBR and are made of transparent materials (fine glass

panels, acrylic or PVC ) conceived for maximum light capture and high densities of photoautotrophic

cells (Brennan and Owende, 2010). Figure 3.5. presents the different types of cultivation systems.

To increase efficiency, PRBs have to be designed to distribute light over a large surface area

in order to provide moderate light intensities for the cells (light dilution), in order to prevent areas

oversaturated with light (photo-oxidation) (Schenk et al., 2008), however photo-inhibition should also

be avoided.

Minimizing auxiliary energy demand is also an important issue. In most PBRs energy is

required for pumping and mixing for the proper mass transfer of CO2 and O2. A good mixing is

necessary, since it homogenizes cell distribution, prevents cell sedimentation, ensures an equal

exposure to light (minimizing self-shading and phototoxicity), and avoids thermal stratification and

nutrient concentration. Also, potentiates gas exchanges, ensuring CO2 supply and strips dissolved

oxygen, hence minimizing harmful effects on microalgae cells (Gouveia, 2011; Kumar et al., 2011).

Pumping energy requirements can be reduced by minimizing the hydrodynamic pressure of the

system. This can be achieved by increasing the diameter of the tubes (something that may also

reduce the overall cost), but they cannot be scaled up indefinitely, otherwise light may not be able to

penetrate the core of the tube (Brennan and Owende, 2010; Chisti, 2007).

Figure 3.5 – Different microalgae cultivation systems (A) raceway pond (B) tubular

PBR (horizontal) (C) tubular PBR (inclined) (D) column PBR (E) flat-plate PBR.

15

Table 3.2. outline the comparative advantages and disadvantages of open ponds and closed

PBR designs.

Table 3.2 - Comparison between the microalgae open cultivation system and closed photobioreactor designs (Brennan and Owende, 2010; Chisti, 2007; Schenk et al., 2008).

3.3.6 Nutritional sources and growth conditions

Microalgae cultivation is the core of microalgae CO2 mitigation strategy. The most relevant

environmental factors influencing microalgae growth and chemical composition include growth

medium (nutrients), light, temperature, pH, salinity, dissolved oxygen, CO2 concentration and toxic

compounds (Rousch et al., 2003). Besides there are biological factors such as the existence of

pathogenic microorganisms and competition with other microalgae (Möller and Clayton, 2007). Finally

reactor operating conditions such as hydraulic retention time, mixing and gas transfer rates are also

important in the growth and cultivation of microalgae.

At the nutritional level, carbon, nitrogen and phosphorous are the macronutrients most

important constituting microalgae cells (Rebolloso et al., 2001).Different microalgae and culturing

strategies toward different compounds. For instance, to produce biodiesel, lipids are the most

Type of system Advantages Disadvantages

Op

en

syste

ms

Raceway pond Cheaper to built Easy to operate Easy to clean Low energy requirements

Difficulty to maintain constant cultivation conditions Contamination issues Requirement of large cultivation areas Poor mixing, light and CO2 utilization Poor biomass productivity

Clo

sed

syste

ms

Tubular photobioreactor

Large illumination surface area Good biomass productivities Suitable for outdoor cultures Control and accurate addition of nutrients and water Relatively cheap

Higher energy consumption Fouling Requires large land space Gradients of dissolved O2, CO2 and pH along the tubes Some degree of wall growth Not demonstrated at commercial scale

Column photobioreactor High mass transfer Low energy consumption Good overall mixing Easy to sterilize Reduced photo-inhibition and photo-oxidation

Small illumination area Sophisticated construction Expensive compared to open ponds Shear stress Only at pilot-scale

Flat plate photobioreactor Large illumination surface area High biomass productivities Easy to sterilize Low oxygen build-up Good light path Suitable for outdoor cultures

Difficult to scale-up Difficult to control temperature Some degree of hydrodynamic stress Some degree of wall growth Only at pilot-scale

16

desirable component; on the other hand, high value health care products rely mostly on proteins and

some fatty acid forms (Campbell et al., 2009).

Apart from carbon, nitrogen is the most important element that is required for microalgae

nutrition. Chlorophyll and protein synthesis being directly associated with their primary metabolism

(Kim et al., 2012). The nitrogen content of the biomass can range from 1% to more than 10% and it

not only varies between different groups (e.g. low in diatoms) but within a particular species,

depending on the supply and availability (Grobbelaar, 2004). Several studies shown that under

nitrogen deficit conditions, microalgae grow at lower rates, but produce significantly more lipids (

Mandal and Mallick, 2009; Chisti, 2007; Illman et al., 2000). Phosphorus is another relevant

macronutrient that has importance on cell growth. Many cellular processes such as energy transfer

(ATP), biosynthesis of nucleic acids (DNA and RNA) and proteins, require phosphorus for

photosynthesis (Agren, 2004). Consequently, limitation growth by phosphate starvation may severely

impact on various aspects, including lipid accumulation and photosynthesis (Wang et al., 2008).

Although microalgal biomass contains less than 1% P, this macronutrient has to be supplied in excess

because phosphates tent to form complexes or precipitations with some metal ions, turning the

phosphate not available to be used by the microalgae (Yun et al., 1997).

In addition to nutrition other operational parameters have to be taken in account. Light

(intensity, source and photoperiod) is a critical parameter for microalgae autotrophic growth (Mata et

al., 2010). However, in excess can lead to photo-oxidation and consequently, a decrease on biomass

productivity (Ho et al., 2012). The light source can be the sunlight or artificial light (Chen et al., 2011).

Temperature and pH are other crucial factors for the cultivation process, ranging between 25º

and 35º and 7 and 9, respectively, for mostly of the species. It is worth to note that usually, the

increase in temperature affects, negatively, the metabolism of CO2 biofixation by microalgae (Zeng et

al., 2011).

Salinity is other parameter that can affect the growth and cell composition of microalgae, both

in open and closed systems. Every microalgae has a different optimum salinity range (Brand, 1984).

In microalgae cultivation, it is also crucial a good mixing, as reported above. On the other

hand, high liquid velocities generates high mixing rates or high turbulence (due to mechanical mixing

or air bubbles) which might cause shear stress and damage microalgae cells (Eriksen, 2008; Möller

and Clayton, 2007), besides requiring a large energy input.

Microalgae growth associated to a flue gas as carbon source, has to be in account toxic

compounds such as heavy metals and gases (NOX, SOX, O2, NH3, etc). Gases such as NOx and SOx

can have significant negative effect towards microalgae growth owing to their hazardous properties

and high toxicity level ( Kumar et al., 2011).

In addition, problems of contamination can arise during monoalgal cultures in open systems.

Common biological contaminants include algae, mould, fungi, virus and bacteria. Contaminants

contribute to an heterogeneous culture, reducing inevitably the biomass production yield, which can

inclusively culminate in the culture loss (Becker, 1994; Borowitzka, 1996).

17

3.3.6.1 Influence of carbon on microalgae growth and biomass quality

Carbon is a key requirement, as the composition of microalgae is about 45%-50% carbon.

Theoretically, for each kg of dry algal biomass, at least 1.65-1.88 kg of CO2 is biofixated (Chisti, 2007;

Doucha et al., 2005).

In aqueous environment CO2 exists in equilibrium with H2CO3, HCO3-, CO3

2- forms which

concentration depends upon pH and temperature. Since exists a fast interconvertible reaction among

them, consumption of any inorganic carbon does not affect the equilibrium.

Different results on microalgae growth can be observed depending on CO2 source. Usual

sources of CO2 for microalgae include atmospheric CO2; CO2 from power plants and industrial exhaust

gases and CO2 chemically fixed in the form of soluble carbonates. In the first route, microalgae has to

be adapted to biofixate between 0,03-0,06% of CO2 from the air, yet it is expected that mass limitation

could slow down the cell growth (Chelf et al., 1993). On the other hand, industrial exhaust gases such

as flue gas or flaring gas, that contains typically 10–20 % CO2 (Tick, 2010), can provide a CO2-rich

source for microalgae cultivation. Nevertheless, the other gases present could be toxic. In the third

route, a number of microalga species is able to use carbonates such as NaHCO3 and Na2CO3 for cell

growth (Huertas et al., 2001; Merret et al., 1996).

The utilization of either CO2 or HCO3− (a predominant form of dissolved inorganic carbon

(DIC), in seawater (pH=8)) are the preferred carbon source for photosynthesis but depending on the

specie. For example, it was shown that growth rate and lipid productivity of Chlorella sp. and

Tetraselmis suecica CS-187 were higher under pure CO2 or flue gas carbon sources as compared

with NaHCO3 (Moheimani, 2012). On the contrary, the growth rate of Thermosynechococcus sp.

increased with increasing DIC. Furthermore, the uptake of CO2 from bicarbonate by photosynthesis

will release hydroxyl anion and increase the pH that can be used as an indicator to confirm the

alkalization process as a result of CO2 and/or HCO3– uptake. The inverse correlation between DIC and

pH may be due to the buffer capacities of bicarbonate, as it is achieved under higher DIC levels and

lead to smaller changes in pH (Su et al., 2012).

CO2 can be tolerated by microalgae cells up to a certain point after which it becomes

detrimental for growth. For certain microalga strains, high CO2 concentrations can be an

environmental stress, which can cause a decrease in biological uptake of CO2 (Sobczuk et al., 1999).

As a rule, biomass productivity increases with the increase of CO2 (v/v) in the gas mixture up to the

threshold percentage, and beyond it the productivity decreases. Experiments realized with Chlorella

sp. T-1 revealed that this strain can tolerate CO2 concentration up to 100% CO2 from a coal fired

thermal power plant; however the maximum growth rate as observed was obtained using 10% (v/v) of

CO2 up to 50% (v/v), with no relevant decrease. It was also concluded that pre-adaptation on lower

percentage of CO2 concentrations increase cell tolerability when after subjected to higher CO2 levels

(Maeda et al., 1995). Furthermore, Scenedesmus sp. could grow under 80% CO2 conditions but the

maximum cell mass was observed in 10% - 20% CO2 concentrations (Hanagata et al., 1992). In a

different study, S. obliquus and C. vulgaris grew well when the culture was supplemented with

enriched air stream contained up to 18% (v/v) CO2 (Morais and Costa, 2007b). However, the best

18

specific grow rates (µ= 0.26 d-1

for C. vulgaris and µ=0.33 d-1

for S. obliquus) were shown when these

microalgae were cultivated with 6% (v/v) CO2 enriched-air. Other microalgae specie, Chlorella

minutissima, could grow under extreme CO2 concentrations [0.035% (v/v) to 100 % (v/v)], strongly

increase the microalgal biomass through photochemical and non-photochemical changes in the

photosynthetic apparatus (Papazi et al., 2008).

Besides, several studies confirm growth limitations when CO2 ambient air is applied to

microalgae systems. For instance, according to Lam and Lee (2013), growth rate of C. vulgaris

increased from 0.156 day−1

to 0.283 day−1

(increment of 44.9%) when the aeration was shifted from air

(0.035%) to air enriched with 5% CO2. In tandem with this, the biomass productivity of C. vulgaris was

significantly increased from 0.315 g L-1

d-1

to 0.727 g L-1

d-1

which correspond to 130.4% increment

(Lam and Lee, 2013). The same was reported by Zheng et al. (2012), particularly suggesting that CO2

concentration above 5% (v/v) may be harmful to microalgal cells and inhibit microalgal growth (Zheng

et al., 2012).

As well as the specific growth rate, biomass concentration and biomass productivities are

improved significantly with the effect of increased CO2 concentrations. For example, S. obliquus SJTU-

3 and C. pyrenoidosa SJTU-2 register an increment of 1.9 and 2.1 times for the best value of

productivity, under 5% (v/v) and 10% (v/v) CO2 levels, compared to air condition, respectively. It was

also reported, that S. obliquus and Spirulina sp. showed good capacities to fix carbon dioxide when

they were cultivated in a controlled three-stage serial tubular photobioreactor, at 30ºC. For S. obliquus,

the specific growth rate and productivity were 0.22 d-1

and 0.14 g L-1

d-1

, with both 6% (v/v) carbon

dioxide and 12%(v/v), respectively, while the maximum concentration was 0.18 g dry cell weight L-1

,

with the highest tested concentration. For Spirulina sp., the corresponding growth rate and productivity

were 0.44 d-1

and 0.22 g L-1

d-1

, respectively (de Morais and Costa, 2007a).

All these observations indicates that supplementing microalgae culture with high concentration

of CO2 can improve their photosynthesis activity, prompting them to reproduce at a faster rate and

attaining higher biomass yield (Lam and Lee, 2013). Beyond that, CO2 optimal growth is strain-

dependent (Zheng et al., 2012).

Apart from biomass growth, the influence of carbon dioxide on biomass quality, namely lipids,

fatty acid composition, pigments, proteins and sugars, play an important role in the economic

valorization (Sydney et al., 2010). Microalgae oils has a high potential for biodiesel production that is

by far one of the most studied biofuel from biomass microalgae and its interest still continues.

Pigments from microalgal biomass have an important application role on cosmetic,

nutraceuticals, pharmaceutical and food/feed industries. The most important pigments include

chlorophylls and the carotenoids such as astaxanthin, beta-carotene and lutein (Spolaore et al., 2006).

Several reports support the idea that relative high CO2 supplies on microalgae cultures can

influence positively the production of lipids, fatty acid composition and pigments (Lam and Lee, 2013;

Tang et al. 2011; Chiu et al., 2009; Chinnasamy, 2009). According to Tang et al. (2011), high CO2

levels (30-50%) could favor high accumulation of total lipids and polyunsaturated fatty acid in S.

obliquus SJTU-3 and C. pyrenoidosa SJTU-2. Highest oil content for each microalgae were achieved

under 50% CO2-enriched air with 24.4 wt.% (S. obliquus SJTU-3) and 26.75 wt.% (C. pyrenoidosa

19

SJTU-2) , while for CO2 ambient condition (0.035%) were attained 15.50 wt.% and 20.9 wt.%,

respectively (Tang et al. 2011). Moreover, the study revealed that an increment on CO2 concentration

could increase the unsaturated degree in fatty acids, since polyunsaturated fatty acids such as

hexadecatrienoic acid (16:3), α-linolenic acid (18:3) and eicosapentaenoic acid (20:5) were high at 30–

50% high CO2 levels. CO2 concentrations in the range of 5 to 20%(v/v) favor the accumulation of lower

unsaturated fatty acids such as hexadecadienoic acid (C16:2) and linoleic acid (C18:2). Lastly, low

CO2 concentrations seemed to be good for the production of saturated fatty acids such as palmitic

acid (C16:0) and short fatty acids (C14:0, C15:0), specially in S. obliquus SJTU-3. The unsaturated,

especially polyunsaturated fatty esters have lower melting points, which is desirable for the

improvement of the low-temperature properties of biodiesel (Tang et al., 2011). Regarding fatty acid

composition, similar trends were observed for C. vulgaris, by Lam and Lee (2013), along the tested

CO2 concentrations [air to 5% (v/v)]. Nevertheless the total lipid content maintained at 18.1–18.7%, do

not changing significantly with CO2 rise. This result were in accordance to some recently reported

studies that a merely 1–6% increment in lipid content in microalgae cells was observed when 10 %

CO2 concentrations are considered (Ho et al., 2010; Yoo et al., 2010).

Concerning pigment content, a C. vulgaris ARC study, recorded at 6% (v/v) CO2 level, at

30ºC, showed that the highest total chlorophyll content (11 mg L-1

) and biomass (0.21 g L-1

) were 60

and 20 times, respectively, more than of the microalga cultured in CO2 ambient air [0.035% (v/v)]. A

sharp increase in chlorophyll synthesis and biomass production were observed, when the CO2

concentration was raised from 1% to 6%. At 16% CO2 level, the concentrations of total chlorophyll and

biomass values were comparable to those at ambient CO2 albeit further increases in the CO2 level,

decay in these two parameters, leading to complete inhibition. In the same study, carotenoid content

was increased twice from ambient CO2 to higher CO2 concentrations [6% (v/v) CO2] (Chinnasamy et

al., 2009).

Among other factors, CO2 concentration is one of the most prevailing factor that contribute to

changes to the microalgae growth and biochemical composition. It is well documented that the

increased concentration of CO2 in the environment may lead to a number of different changes in

physiological as well as fine structure properties of cells (Tukaj et al., 2007).

3.3.6.2 Influence of CO2 aeration rate on microalgae growth and biomass quality

Aeration rate is a key parameter in mass transfer of CO2. This particular parameter is very

important when seeking to achieve high-productivity microalgae bio-mitigation of CO2 from input

gases. Even more, supply of CO2 and its transfer to medium culture was flagged as one third of the

total cost of the production of microalgal biomass in the largest scale systems (Sobczuk et al., 1999).

It can be thought that productivity could be decreased when low CO2 aeration rates are

applied to a microalgae culture. However, simply increasing CO2 aeration rates does not necessarily

lead to a higher CO2 fixation efficiency (Li et al. 2011). It was shown increasing aeration rates from 0.1

vvm to 0.5 in S. obliquus WUST4 culture medium resulted in decreasing CO2 removal efficiency from

67 to 20% (Li et al., 2011). In fact, supplying high CO2 concentrations with low gas flow rate leads to a

20

low inorganic carbon loading in the liquid phase and a low concentration of DIC. Thus, some

microalgae can tolerate high CO2 concentrations with low gas flow rates (Raeesossadati et al., 2014).

Therefore, low aeration rates favor bigger gas retention time in the culture medium, leading to an

increased interface between CO2 and microalgal cells. The influence of bubble coalescence could be a

significant factor; as it increases with increased flow rates, larger bubbles raise to the surface at a

faster speed than smaller bubbles and the bubble area per unit of gas volume declines. The CO2

assimilation by microalgal cells can diminish (Chiu et al., 2009).

The opposite is also reported, where increasing aeration rates improved CO2 removal rates.

For instance, the effect of aeration rates from 0.25 to 0.50 vvm improved significantly the CO2 fixation

rate on Chlorella sp MT-7 and MT-15 (Ong et al., 2010).

Additionally, the effect of different aeration rates (0.001, 0.002, and 0.005 ms−1

) on the dry

weight for C. vulgaris and D. tertiolecta was assessed. The maximum biomass concentration was

obtained for both C. vulgaris and D. tertiolecta under the highest gas flow rate (0.005 ms−1

) at 12%

CO2 with 3.79 and 3.17 g L−1

, respectively (Hulatt and Thomas, 2011). Another experiment (Singh et

al., 2015), indicated that more pronounced CO2 aeration rates (from 0.04 to 0.2 vvm ) lead to increase

on microalgae growth rate, albeit further increase (for 0.2 vvm), growth rate declined. While it is true

that decreasing aeration rates lead to CO2 biofixation efficiency increase, the opposite results have

being referenced, supporting the idea that improved mass transfer can occur at high gas flow rates.

Several production parameters (temperature, light, initial concentrations, PBRs design, nutrient

availability, etc) might arise as consequence for so much divergent results (Hulatt and Thomas, 2011),

despite the intrinsic strain-dependent physiological responses.

Regarding biomass quality, very little studies were performed specifically to determine the

impact of CO2 aeration rates on biomass quality (Anjos et al., 2013; Zheng et al., 2012). Anjos et al.

(2013) tested C. vulgaris P12 under 2%, 6% and 10% (v/v) CO2-enriched air with different CO2

aeration rates (0.1 vvm, 0.4 vvm, 0.7 vvm). The content of starch, proteins and lipids of microalgae

showed statistically no significant differences along aeration rates tested under the diverse CO2

concentrations. The study suggests that such behavior could be explained by the moderate conditions

implied to the culture (Anjos et al., 2013). Similar results were obtained by Zheng et al. (2012),

although lipid accumulation is perceived when aeration rate was increased from 0.1 to 0.5 vvm, but

lipid production seems to be discouraged after this point. Particularly regarding fatty acid composition,

high content of unsaturated fatty acids is presented under the highest flow rate (2 vvm, under 5% (v/v)

CO2 concentration] , what is suitable for the production of good-quality biodiesel (Zheng et al., 2012).

3.3.7 Microalgae harvesting and downstream processing

A good harvesting method is an essential step to ensure the high-quality of the biomass,

however this challenging step is extremely expensive and energy-intensive (Wang et al., 2008), as it

accounts for 20-30% of the total cost of production (Carlsson et al., 2007). Main difficulties in

harvesting microalgae include low cell densities (in the range of 0.3-0.5 g L-1

), coupled with the small

size of cells (typically in the range of 2-40 µm) and negatively charged surface. The principal

21

techniques used to harvest biomass encompasses centrifugation, flocculation, membrane filtration,

gravity sedimentation, flotation, foam fraction, ultrasonic separation, electrocoagulation (Brennan and

Owende, 2010). Microalgae cell immobilization has been proposed to circumvent the harvesting issue,

but large-scale applications are limited (Mallick, 2002). Electrocoagulation is an emerging technology,

providing good efficiency, low cost, fast operation, almost null contamination of the biomass nor

changing its composition (Matos et al., 2013).

Selection of the harvesting method depends upon the strain properties (density, size,

conductivity, pH of the culture medium), the main products desired, since certain species are much

easier to collect than others and the importance or not of biomass contamination (Olaizola, 2003).

Generally, microalgae harvesting involves a two-step process - the bulk harvesting - aiming

the separation biomass from the bulk suspension. Total solid matter achieved can reach 2-7% using

the flocculation, flotation, electrocoagulation or gravity sedimentation methods. These methods

discriminate on a size and density basis in performing the biomass separation. The second stage is

thickening, aiming the concentration of the slurry by centrifugation, filtration and ultrasonic aggregation

– being more intensive in energy requirements (Brennan and Owende, 2010).

After harvesting microalgae biomass slurry has to be dried/dewatered and cell disrupted

depending on the final product required.

Cell disruption is often required for recovering intracellular products from microalgae. In this

approach is better percolation of solvents or fluids though the cell to improve extraction efficiency

(Williams and Laurens, 2010). Lipids, proteins or internal metabolites can be recovered from biomass

by means of physical processes (e.g., cold press, bead milling, ultrasonic) with solvent extraction.

Solvents (n-hexane, acetone, chloroform, etc.) are widely used to extract fatty acids and metabolites

such as pigments (e.g., beta-carotene, lutein and astaxanthin) from microalgae biomass (Grima et al.,

2003). Nevertheless, in the last decades, to ensure an increase in extraction efficiency and reduce

toxic organic solvents supercritical fluid extraction has been used (Nobre et al., 2013; Mendes et al.,

2003).

3.3.8 Applications of the microalgal biomass

The biomass that was produced by “cleaning” gases and wastewaters have an important

biotechnological potential for a huge number of applications. From animal feed, human food and

nutraceuticals to cosmetic and pharmaceutical industries, there is a wide range of commercial

possibilities. Note, however, that the quality of the flue gas and wastewater might hamper specific

applications in the medical and food field (Kumar et al., 2010). Industrial chemicals extracted from

microalgae include: glycerol, which is widely employed in food and personal care products; fatty acids,

used in cosmetics; astaxanthin, beta-carotene and lutein used as antioxidants and anti-inflamatory for

therapeutics and coloring agents in food, cosmetics and aquaculture; poly-b-hydroxybutyrate, used in

bioplastics; and polysaccharides, such as agar, alginates and carrageenans, which are employed as

thickening agents for foods (Pulz and Gross, 2004). Moreover the biomass stores a great amount of

22

energy that can be converted into biodiesel (by transesterification of the oils) (Gouveia and Oliveira,

2009), bioethanol (by alcoholic fermentation of the sugars) (Miranda et al., 2012), hydrogen

(photobiologically or by dark fermentation) (Batista et al., 2014; Ferreira et al., 2012), biogas (by

anaerobic digestion) (Batista et al., 2014), syngas, methane, butanol and jetfuel by thermochemical

technology (e.g. combustion, gasification, pyrolysis) (Brennan and Owende, 2010; Ho et al., 2011a).

Likewise, microalgae biomass when subjected to some conversion technologies (pyrolysis,

fermentation or anaerobic digestion) result in solid residues that can be potentially applied in

agriculture or horticulture sector as a fertilizer (Brennan and Owende, 2010).

23

4. Materials and methods

4.1 Microorganism

The microalgae species used for this experiment were Scenedesmus obliquus (S. obliquus),

strain ACOI 204/07, obtained from the Algae Collection from Coimbra’s University (Portugal); Chlorella

protothecoides (C. protothecoides), strain 25, provided by UTEX Collection (Texas University of

Austin, USA) and Chlorella vulgaris (C. vulgaris), strain 58, isolated by INETI (former LNEG, Lisbon,

Portugal).

4.2 Growth medium composition

Each microalga specie had a proper culture medium. Bristol’s Medium (Vonshak, 1986) was

used to grow S. obliquus (Table 4.1) and Chlorella Medium (Starr and Zeikus, 1987) was used to grow

both C. vulgaris and C. protothecoides (Table 4.2).

Table 4.1 – Bristol Medium composition.

Chemical formula Purity Brand Concentration(g L-1

)

NaNO3 99% Riedel de Haën 0.250

KH2PO4 99% Panreac 0.175

K2HPO4 99% Panreac 0.075

MgSO4.7H2O 99% Merk 0.075

Fe-EDTA 12-14% Sigma-Aldrish 0.060

CaCl2.2H2O 99% Riedel de Haën 0.033

NaCl 99.5% Panreac 0.025

Trace solution element - - 1 mL L-1

Table 4.2 - Chlorella Medium composition.

Chemical formula Purity Brand Concentration (g L-1

)

NaHCO3 99.7% Panreac 0.50

KH2PO4 99% Panreac 1.25

KNO3 99% Riedel de Haën 1.25

MgSO4.7H2O 99% MERCK 1

CaCl2.2H2O 99% Riedel de Haën 0.11

Fe-EDTA solution - - 10 mL L-1

Trace solution element - - 1 mL L-1

24

Table 4.3 - Trace solution element composition.

The media were previously sterilized by autoclaving (at 121ºC, 2 bar over 20 min) to avoid

contamination during culture process. In the Chlorella Medium it was necessary to sterilize on different

Erlenmeyer’s the KH2PO4 and Na2HCO3, from the other components, because the phosphate reagent

tent to precipitate.

4.3 Inoculum preparation

Both Scenedesmus and Chlorella autotrophic cultures (pre-inoculum) were grown on 800 mL

Bristol Medium and Chlorella Medium, respectively, in 1L Erlenmeyer flasks and picked up every

month and a half. The media were previously sterilized in an autoclave (88 AGC, Uniclave, Portugal)

at 121ºC, 2 bar for 20 min. The flasks were kept in an orbital incubator at temperature around 28ºC,

under continuous fluorescent light (7.6-9.1 μmol m-2

s-1

). After four days of incubation, the pre-

inoculum was transferred to the inoculum flask and then to the 1L bubble column photobioreactor

(PBR). The manipulation was done by flame to maintain aseptic conditions. The inoculum volume was

based on the optical density (OD) (λ = 540 nm) of the pre-inoculum. It was diluted to obtain an OD

roughly of 0.090 and then transferred to the photobioreactor totaling 1L of culture. All experiments

were made in duplicate.

4.4 Microalgae cultivation

4.4.1 Photobioreactor assembly

The culture growth was carried out on 1 L of microalgae culture in a glass bubble-column

PBR. The photobioreactor was composed by an entrance with two tube branches: one to the entrance

of the CO2 enriched-air linked to a cotton filter, providing agitation, and other, to collect samples from

time to time to measure the OD, pH and cell dry weight. Finally, it has another cotton tube out with a

filter, to air flow (Figure 4.1A). The PBR was previously sterilized by autoclaving (121ºC, at 2 bar, for

Chemical formula Purity Brand Concentration

(mg L-1

)

H3BO3 99.8% MERCK 286

MnSO4.4H2O 99% Riedel de Haën 203

ZnSO4 99% BHD Chemicals 22

CoSO4.7H2O 99% T.J. Baker Chemicals 9

Na2MoO4.2H2O 97% MERCK 6

CuSO4 99% MERCK 5

25

20 minutes) and filter tubes were sterilized in a dry oven (Schutzart DIN 40050-IP20 Memmert), at

180ºC, for 40 minutes.

Figure 4.1 - (A) Bubble column PBR assembly (1 – Inlet air flow; 2 – Output to collect samples; 3 – Output air

flow; 4 – Inoculum flask); (B) PBRs during microalgae cultivation experiments

4.4.2 Cultivation Conditions

Each PBR was operated on batch mode, approximately at 28ºC of temperature, with an

external light source provided by three racks of fluorescent lamps (TL-D 18 w/54-765, Philipps Co.) of

18 Watts, placed on the both sides (Figure 4.1B). Cultures were subjected to 5,48 klux (74 μmol m-2

s-

1) of light intensity measured at the surface of the PBR by Lux Meter (PHYWE), in a continuous mode.

Agitation and aeration was provided by bubbling CO2-enriched air through a needle (inner diameter of

0.8 mm) at the bottom of the PBRs. Different CO2 concentrations were used in the experiments for the

three microalgae species with a feeding rate of 1 vvm (volume gas per volume media per minute). In

the Table 4.4 is present the CO2 concentrations studied in this work for each microalgae.

Table 4.4. CO2 concentrations (v/v) tested in C. vulgaris, C. protothecoides and S. obliquus.

% CO2 (v/v) concentration Microalgae specie

S. obliquus C. vulgaris C. protothecoides

0.035% (Air) Yes Yes Yes

2.5% Yes No No

5.0% Yes Yes Yes

7.5% Yes No No

10% Yes Yes Yes

15% Yes No No

26

The gas flow rate was regulated by a system of tweezers and adjusted with a flow meter

(model GMF17, Allborg, USA). The gas composition required for each experiment was regulated by a

gas mixer (model Map Mixer 9001 ME, PBI Dansensor, Denmark) coupled to 10 L pressure/buffer

tank – directly mixed bottled CO2 (99.9% purity) with compressed atmospheric air unto defined CO2

proportion (Figure 4.2).

The correlation between the real CO2 in the mixer and the measured CO2 by the sensor, was

previously established by the proper calibration curve (Annex I).

To perform the experiments with different CO2 aeration rates, it was selected S. obliquus and

the same conditions above were employed (T≈ 28ºC; continuous light intensity = 5.48 klux), except for

5% (v/v) CO2 concentration where aeration rates tested were 0.25, 0.50, 0.75 and 1 vvm. The control

of gas flow rate was regulated with the same tweezers system and adjusted by the flow meter. The

inlet and outlet air was monotorized by a CO2 sensor (model Checkmate II, PBI Dansensor).

4.5 Determination of growth kinetic parameters and CO2 fixation rate

For the three species, the growth kinetic parameters were accessed in order to understand the

influence of CO2 concentration on microalgae growth. The parameters evaluated were the specific

growth rate (µ), the average productivity (Pa) and the theoretical CO2 fixation rate (PCO2). The specific

growth rate (day-1

) was calculated according with the Equation 1 (Abreu et al., 2012):

Where X0 and Xt are the dry weight cell concentrations (g L−1

) of cells at beginning (t0) and at

the end of exponential growth phase (t1) represented in day-1

.

To calculate the average biomass productivity (Pa), dry weight cell concentration was used (X,

g L−1

) and was based on the Equation 2:

µ (d 1 = ln 1 – ln 0

t1– t0 1

g L 1 = – 0

t – t0 2

Figure 4.2 - Schematic diagram of the experimental setup.

27

X0 and Xt were the dry weight cell concentration (gL−1

) at beginning (t0) and at the final day of

the culture growth (tt) (day-1

).

The maximal theoretical CO2 fixation rate (PCO2) was calculated based on the Equation 3

(Tang et al., 2011)

CO2 g L

1 d 1 3

Where Pmax denotes the maximum biomass productivity (g L-1

d-1

) that was calculated based

on Equation 2 (from a daily estimation it was chosen the highest value of productivity). 1.88

represents the theoretical value of CO2 biofixated in grams per gram of biomass produced, assuming

that derived from a mass balance with the typical molecular formula of microalgal biomass

CO0.48H1.83N0.11P0.01 (Chisti, 2007b).

For the experiment with different flow rates (S. obliquus 5% (v/v) CO2 enriched-air) it was

estimated the real CO2 biofixation rate (RCO2, g L-1

d -1

) (Equation 4). It was used a CO2 sensor (model

Checkmate II, PBI Dansensor, Denmark) to measure the inlet and outlet CO2-flow and the calculation

take into account the conversion of vvm to Liter of air per Liter of culture spent in one day that is given

by the Equation 5.

a (Ld-1) = b (LL-1min-1) × 60 (min) × 24 (h) 5

Where b corresponds to the volume of air entering per one Liter of culture per minute which

multiplied by 60 and 24, it is achieved the volume of air entering through the cultures per day (a).

The estimation of the CO2 amount that enters for the bubble-column (CO2 in) is given by the

Equation 6:

Where corresponds to the amount of air entering in the bubble-column, % CO2 is the

percentage of CO2 imposed to the air current flow and 1.98 corresponds to the density of the CO2 (g L-

1).

The estimation of the CO2 outlet flow (CO2 out) is given by Equation 7:

Where c corresponds to the average percentage of the CO2, read in the outlet flow air (on the

flow meter) during the cultivation process; d corresponds to the percentage of CO2 read in the inlet

flow air.

RCO2 (g L-1 d -1) = CO2 in – CO2 out 4

CO2 in (g L-1 d -1) = (Ld-1) × % CO2 × 1.98 6

7

28

4.6 Analytical methods

4.6.1 Determination of microalgal biomass growth

Culture samples were collected from the bubble-column PBR at defined time intervals (in

general daily or 48 hours). The growth was evaluated by measuring the optical density (OD) and

determining the dry cell weight (DCW).

The OD was analyzed using the UV/VIS spectrophotometer (model U-2000, Hitachi, Tokyo,

Japan) at wavelength of 540 nm (denoted ODλ540), after proper dilution with filtered water (Millipore), to

range an OD between 0.1-0.9. The pH was also measured daily using an electrode (inoLab level 1).

The DCW of microalgae biomass was obtained filtering by vacuum 10-20 ml aliquots of culture

through a pre-weighed glass microfiber filter paper Whatman GF/C of with 0.45 µm of pore size and

0.47 mm of diameter (Whatman International Ltd., UK). Filters were washed with Millipore water and

then dried in an oven at 80ºC overnight, until the weight was constant (approximately 4 h). Both

weightings were performed in a Mettler Toledo classic AB204-S scale. The dry weight of the blank

filter was subtracted from that of the loaded filter to obtain the microalgae dry cell weight. The

maximum DCW (DCWmax) corresponded to the highest value obtained by the method described

above.

4.6.2 Lipid extraction and analysis of fatty acid profile

After the appropriate cultivation, the cells were harvested from the culture by centrifugation

(Figure 4.3A,B) at 10000 rpm and 4ºC, for 10 minutes (model Heraeus Multifuge 3S R+, Thermo

Scientific, Whaltman, USA) and collected to Petri dishes (Figure 4.3C) with a rubber spatula. The

biomass was dried using a freeze-dryer (Heto PowerDry LL3000) prior to downstream processing,

such as lipid and pigment extraction.

Lipid extraction was performed in a test tube, for each sample in duplicate, using the Lepage

and Roy method (Lepage and Roy, 1986). The method consists on a direct transesterification to fatty

acid methyl esters (FAMEs). The dried biomass was weighed (aliquots about 100 mg) and was added

Figure 4.3 - (A) Sample to centrifuge; (B) Sample after

centrifugation; (C) Biomass collected to the Petri dish.

29

2 mL of the mixture methanol/ acetyl chloride solution (95:5 v/v) previously prepared and maintained in

an ice bath. Then, 200 µL of an internal standard solution C17:0 (5mg/ mL; Nu-Check-Prep, Elysian,

EUA) in petroleum ether 80-100ºC was added to the tube with the mixture. The tubes were maintained

under nitrogen atmosphere, closed and heated in a water bath at 80ºC for 1 hour in the dark. After

cooling down to room temperature, 1mL of n-heptane (p.a., MERCK) and 1 mL of distilled water (to

facilitate phase separation) was added to extract methyl esters. The superior (organic) phase was

filtrated within cotton filter with an anhydrous sodium sulfate bed and transferred to vials under

nitrogen atmosphere (to prevent oxidation of lipids).

The lipid profile was analyzed immediately by GC-FID – gas chromatography - (Scion GC-

436, Bruker, Germany) equipped with SUPELCOWAX 10 (Supelco, Bellafonte, Palo Alto, California,

USA capillary column (0.32 mm of internal diameter and 0.25 μm of film thickness . The carrier gas,

He, was kept at a constant rate of 1.6 mL/min. The column was programmed at an initial temperature

of 200ºC over 20 min, then increased at 2ºC min-1

to 220ºC and held over 14 min. The injector and

detector temperatures were 250 and 280°C, respectively, and split ratio was 1:20 for 5 min and 1:10

for the remaining time. The column pressure was 13.5 psi.

FAMEs were identified by comparison of the retention times of the samples with that of

standard FAMEs mixture. Fatty acid composition was calculated as a percentage of the total fatty

acids present in the sample, determined from the peak areas (Equation 8):

Where Am.e is the area of the each identified methyl ester; ƐA is the sum of the total peak

areas; Api is the area corresponding to the internal standard (C17:0).

The final oil content was calculated according the Equation 9:

Where ƐA is the sum of the total areas; Api is the area corresponding to the internal standard

(C17:0); mpi is the mass of internal standard and msample (mg) is the mass of the microalgae sample.

4.6.3 Total pigment (chlorophyll and carotenoids) extraction and analysis

In a centrifuge tube (HACH) was weight 10 mg of microalgae dried sample and were added

roughly 0.7 g of glass beads and 2 mL of acetone (90%). The tube was involved by aluminum foil (to

protect against light and prevent pigment oxidation), then was vigorously homogenized in a vortex

over 2 min and transferred to an ice bath for more 2 min. The last step was repeated twice, then the

8

9

30

mixture was centrifuged (Sigma Sartorius 2-6E) at 3900 rpm over 10 min and the extract was

recovered to another tube. The pellet was again resuspended in acetone, vortex, subjected to the bath

ice and centrifuged. This procedure was repeated the necessary times until the extract become

colorless.

Absorption spectra of the acetone extracts were measured in a spectrophotometer (Hitachi

2000) reading between 380 and 700 nm.

The quantification of total chlorophyll content was estimated based on Ritchie's Equations

(2008) (Equations 10, 11):

Where A630 is the absorbance at 630 of wavelength; A647 is the absorbance at 647 of

wavelength A664 is the absorbance at 664 of wavelength; A691 is the absorbance at 691of wavelength.

Since the value of the total chlorophyll (Ca+b) is the sum of chlorophyll a and chlorophyll b, the

total chlorophyll content (Ct) can be estimated by the Equation 12 :

Where Vf is the final volume of the extract and Vi is the initial volume of the extract (before

centrifugation).

The pigments in the sample extract’s were identified by TLC (Thin Layer Chromatography)

using a silica gel plate, that was previously activated by heating in an oven at 80ºC for 30 min. The

eluent consisted in a mixture of petroleum ether 40-60ºC, acetone and diethylamine in a proportion of

10:4:1 (v/v/v).

The identification and quantification of the pigments on the extracts were also performed, on a

Hewlett Packard HP-1100 series liquid chromatograph (Hewlett Packard, Waldrom, Germany). The

HPLC was equipped with a Vydac C18 TP54 reversed-phase column (250×4.6 mm μ-bondapack) and

a UV–Vis detector, set on the wavelength relative to the maximum of absorbance found for each

pigment (450 nm for lutein and beta-carotene and 600 nm for chlorophyll a). The eluent used was

methanol (100%). The pigments were eluted over 20 min with a flow rate of 1 ml/min with a column

pressure of 52 bars. Before the injection all extract samples were filtrated through a PTFE filter syringe

(Membrane solutions), with 0.22 mm of pore size and 13 mm of diameter to a vial.

Each pigment was identified through the comparison of retention times of the standards

(chlorophyll a (>90%, Wako Pure Chemical Industries), beta-carotene (95%, Sigma-Aldrish), lutein

(≥95 , Extrasynthese . Quantification of these pigments, namely beta-carotene and lutein, in the

extracts was performed using calibration curves of external standards (Annex II and III). Standard

10

11

12

31

solutions of beta-carotene and lutein with concentrations in the range 0.08-3.78 mg L-1

and 0.12-11.55

mg L-1

, were used respectively.

32

5. Results and discussion

5.1 Influence of CO2 concentration on microalgae growth

S. obliquus, C. vulgaris and C. protothecoides were cultivated over 15 days in batch mode, at

approximately 28ºC, under 74 µmol m-2

s-1

of light intensity, on Bristol’s Medium for S. obliquus and

Chlorella’s Medium for C. vulgaris and C. protothecoides. Different CO2 concentrations were used,

such as 0.035% (v/v) (Air), 5% (v/v) and 10% (v/v) of CO2 with a feeding rate of 1 vvm. For S.

obliquus, other CO2 concentrations were also used [2.5% (v/v), 7.5% (v/v) and 15% (v/v)]. To an

easier comparison between the results, the data regarding all concentrations for the three algae will be

presented together.

Growth kinetic parameters (specific growth rate and average productivity), maximum dry cell

weight and theoretical CO2 biofixation rate were assessed, to understand the growth performance and

the influence of CO2 concentration on growth.

5.1.1 Optical density (OD)

The growth curves were obtained applying the logarithmic scale on the OD readings at 540

nm, over the cultivation time for all experiments. Figure 5.1 presents the growth behavior of

microalgae species under different CO2 concentrations.

S. obliquus cultures shown a better performance when CO2 was present, while maintaining a

similar growth pattern. However, a slightly better performance was detected for cultures under 2.5%

and 5% (v/v) CO2 concentrations. As it was expected, S. obliquus culture under air conditions

presented the lowest performance, since do not have an additional CO2-enrichment feed as carbon

source. It can also be notice that S. obliquus seem to have good tolerance under air plus 15% (v/v)

CO2, do not demanding any sort of pre-adaptation. It is well documented that pre-adapting cells at a

low concentration of CO2 is an alternative approach to increase CO2 tolerance without effects during

microalgal growth (Lee et al., 2002). Chlorella sp. cells that were pre-adapted to 2% (v/v) CO2 not only

grew to a high-density microalgal culture but also grew fast at 10% (v/v) or 15% (v/v) CO2 (Chiu et al.,

2008).

Since microalgae use CO2 as a nutrient source, it was expected that growth with higher CO2

concentration in the gas-mixture, would increase their growth performance, however, S. obliquus

seems to prefer CO2 intermediate levels: 2.5% (v/v) and 5% (v/v) to grow. This was reported by

several researchers who sorted out that Chlorella sp., Nannochloropsis oculata, Dunaliella terticlecta

and S. obliquus had optimal growth potential in the range of 2–6% CO2, and the growth was

decreased by increasing CO2 levels (Tang et al., 2011; Fulke et al., 2010; Chiu et al., 2009). In this

work S. obliquus had a good results on growth at 2.5 and 5% (v/v) CO2 (fitting on that range) but also

33

had a good development under higher CO2 concentrations. C. vulgaris growth is also described in

Figure 5.1 and it is observed that this microalga specie achieved a better growth under 10% (v/v) CO2

concentration. However, it seems that take more time to boost the exponential phase. Apparently,

higher CO2 concentrations on the culture, lead to a longer adaptation which can be represented by a

pronounced lag phase. Cultures under air condition exhibited better growth than 5% (v/v) condition,

which was not expected. Room air containing around 0.04% of CO2 could not meet the need of

microalgae photosynthesis on carbon source (Chiu et al., 2008). Besides, a growth response study

varying concentrations of carbon dioxide (ranging from 0.036 to 20%) using C. vulgaris ARC1 strain,

shown that after the 2nd day of incubation the best increase in biomass was recorded for 6% of CO2-

enriched air (Chinnasamy et al. , 2009). So it was expected that in this study, the intermediate level of

CO2 [5% CO2 (v/v)] achieved better results. It can be due to a different strain used and/or probably

other culture conditions established.

C. protothecoides growth curve is represented in Figure 5.1 and evidenced better growth

under 10% (v/v) CO2 concentration, followed by air and thereafter by 5% (v/v) CO2. It was verified that

10% (v/v) CO2 enriched-air potentiates a good growth performance, meaning a superior adaptation by

C. protothecoides to high CO2 levels.

Figure 5.1 - Growth curves of the cultures of Scenedesmus obliquus, Chlorella vulgaris and Chlorella


s-1

; 1vvm) subjected to different CO2 concentrations tested: [Air ( );

2.5% CO2 ( ); 5% CO2 ( ) 7.5% CO2 ( ); 10% CO2 ( ); 15% CO2 ( )]. Vertical bars represent standard

deviations among the average of two duplicates.

34

The pH of the culture was regularly monitored during the cultivation process. S. obliquus

cultures presented variations roughly in the range of 5.50-7.70, except for the culture subjected to air

conditions, which suffered a pronounced fluctuation ranging from 6.50 to 10.50. For C. vulgaris, pH

media ranged between 6.61 and 10.17, except for the culture supplied with 5% (v/v) CO2

concentration (6.56-7.98). C. protothecoides media showed a behavior very similar to S. obliquus

media, varying in similar ranges, except once again for the air conditions (6.76-10.53).

The similar fluctuations presented both for S. obliquus and C. protothecoides, for CO2-

supplied cultures, could be explained by the increased dissolved inorganic carbon (DIC), as

bicarbonate in the medium. In this case bicarbonate can act as buffer, as it is achieved under higher

DIC levels, can result in smaller pH changes and in some cases become the culture medium slightly

acidic (Su et al., 2012). On the other hand, the results for air conditions could be explained for the

lower concentrations of CO2 on the culture, meaning lower dissolved inorganic carbon in the medium

culture. This can result in higher pH and therefore, medium alcalinization. For C. vulgaris the pH

variance for air and 5% CO2 concentration can be explained by the same reasons pointed to S.

obliquus and C. protothecoides. However, the pH result for 10% (v/v) CO2 concentration does not

follow the logic stated above, since the fluctuation was high. The alcalinization of the medium could be

explained by a strain-specific response to the higher CO2 concentrations (Baba and Shiraiwa, 2007).

5.1.2 Maximum dry cell weight (DCWmax)

Figure 5.2 presents the DCWmax (g L-1

), determined according to the Section 4.6.1., for all

microalgae species and CO2 concentrations tested.

Figure 5.2 – Representation of the maximum dry cell weight (g L-1

) of the cultures of S.


s-1

;1 vvm)

subjected to different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v)

CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical bars

represent standard deviations among the average of two duplicates.

35

The DCWmax ranged between 0.9 and 5.8 g L-1

, among the different tested microalgae

species. The highest DCWmax was attributed to C. protothecoides [10% (v/v) CO2] with 5.8 g L-1

(day

15), whereas S. obliquus and C. vulgaris obtained a maximum growth of 3.4 g L

-1 (day 12) and 1.8 g L

-

1 (day 15), respectively, both for [(10% (v/v) CO2)]. Therefore, for all studied microalgae, the maximum

dry biomass growth was attained under 10% (v/v) CO2 concentration. Despite of the differences

between the values, it is positive that higher amounts of biomass are produced in relatively high CO2

concentrations.

Compared to the literature, Scenedesmus apparently produced 2 times more biomass than

other algae, in similar conditions (Tang et al., 2011). On the other hand, C. vulgaris shown higher

DCWmax, approximately 1.3 times more than the study reported under the same CO2 concentration

(Zheng et al., 2012). In fact, results from Zheng et al. (2012) are very similar to the highest value for

C. vulgaris of this study for 5% (v/v) CO2 concentration. However, it varies according the strains

(Zheng et al., 2012).

The best result achieved for C. prothotecoides (5.8 g L-1

) resulted of an increase almost about 4

times when compared to the control condition (1.8 g L-1

) and with the one reported by Santos et al.

(2011) [2 g L-1

, under 0.035% (v/v)]. C. protothecoides could be a good potential candidate to mitigate

CO2, producing high amounts of biomass, and could contribute to several applications, such as animal

feed, fertilizers and/ or bio-energy production (Miranda et al., 2012).

5.1.3 Specific grow rate (µ)

The specific growth rate (µ), expressed in d-1

, is a very important parameter to evaluate how

fast the cells are dividing in a culture. Analyzing the Figure 5.3, the values for µ ranged from 0.32 to

0.99 d-1

. The highest value was achieved by Scenedesmus at 7.5% (v/v) CO2 concentration (µ = 0.99

d-1

), followed by the cultures with 10% (v/v) (µ = 0.92 d-1

) and 5% CO2 (µ = 0.92 d-1

) concentrations.

Figure 5.3 - Representation of the specific growth rate (d-1

) of the cultures of S. obliquus,

C. vulgaris and C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

; 1vvm), subjected to

different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( );

7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical bars represent

standard deviations among the average of two duplicates.

36

The S. obliquus growth seems to be favored by intermediate CO2 levels. In fact, literature

reports similar specific rates achieved under proximate CO2 concentrations (µ = 0.94 d-1

) for 5% (v/v)

CO2 (Tang et al., 2011). Both C. vulgaris (µ = 0.48 d-1

) and C. protothecoides (µ = 0.72 d-1

) do not

achieve values as high as S. obliquus. However, the highest results for both Chlorella were registered

under 10% (v/v) CO2 concentration, in which the growth rate was accelerated, compared to bubbling

with air. It is important to highlight that low µ, for intermediate CO2 levels [5% (v/v)] (for both Chlorella),

were not expected. In somehow, cultures seemed to have an inhibition growth and for this reason,

experiments should be repeated. For instance, Lam and Lee (2013) found that C. vulgaris grows

favorably under 5% (v/v) CO2 concentration, with an increment of 44.9%, when the aeration is shifted

from air (0.035%) to air enriched with 5% (v/v) CO2. The same is corroborated by Zheng et al. (2012)

that reported better specific grow rates (µ = 1.20 d-1

) for C. vulgaris cultures, under 5% (v/v) CO2

enriched-air.

Despite the lower specific growth rates (compared with S. obliquus), both Chlorella seem to

have potential to grow under high CO2 levels [10% (v/v) CO2] what could be positive in terms of CO2

mitigation from flue gases. Likewise, S. obliquus can be a possible candidate for establishing a CO2

mitigation strategy since it presents the best performance and uniformity in terms of growth specific

rates along the tested CO2 concentrations.

5.1.4 Average productivity (Pa)

The average biomass productivity (Pa), expressed in g L-1

d-1

, was also evaluated. Pa is

depicted in Figure 5.4 and exhibits S. obliquus as the microalgae that reached the better results.

Figure 5.4 - Representation of the average productivity (g L-1

d-1

) of the cultures of S.


s-1

; 1vvm),

subjected to different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v)



37

The higher values were achieved for 2.5, 7.5 and 10% (v/v) CO2 levels, being 0.37, 0.34, 0.35

g L-1

d-1

, respectively. The average productivity for this microalga far exceeded the culture control

(more than the double, for all tested CO2 levels), meaning a great positive influence of CO2 during

growth. In comparison with other studies, Pa, obtained from S. obliquus, average productivities

surpassed in 2-3 times the referenced values (Tang et al., 2011; Morais and Costa, 2007).

C. vulgaris presented better productivity for the control culture (0.15 g L-1

d-1

) wherein cultures

subjected to higher CO2 concentrations reached lower values. According to the literature, for similar

CO2 concentrations, this microalga (C. vulgaris) achieved lower productivities (3-7 times) than the

present study (Lam and Lee, 2013; Zheng et al., 2012). This probably reflects a different adaptation of

the microalga to the experimental conditions and a possible strain-dependency.

For cultures under air addition, different productivities were observed. Zheng et al (2012)

observed 4.8 times more biomass productivity than in this study, on the contrary, Lam and Lee (2013)

4.9 times less. The opposite results could possibly be related with different initial concentration of

biomass, used in those studies and/or several other culture parameters.

C. protothecoides showed good results in terms of biomass productivity (0.24 g L-1

d-1

),

specially when the gas mixture is composed by 10% (v/v) of CO2. It is possible to register an

increment, (about 1.5 times more) when air and 10% CO2 conditions are compared. Once again, low

values of productivities are noted, for 5% CO2 levels, as well as for C. vulgaris, meeting the lowest

specific growth rates found in this study Figure 5.3.

S. obliquus seems to be the more appropriate specie (among the ones tested) in these

regards, since productivity is economically important for the overall microalgal CO2 biofixation process,

not only due to attaining more microalgal biomass for commercial uses but also due to shortening the

cultivation time, and the obvious environmental benefits.

5.1.5 CO2 biofixation rate

Theoretically, the higher CO2 biofixation rate the higher assimilation of carbon in

photosynthesis and inherently the higher mitigation of CO2 from the gas stream. The theoretical CO2

biofixation rate, expressed in g CO2 L-1

d-1

, was calculated according to the Equation 3 on the

Section 4.5 and the results are shown in Table 5.1

38

Table 5.1. - Theoretical CO2 biofixation rates between the studied microalgae strains, according to the

percentage (v/v) of CO2 concentration tested.

S. obliquus presented a good biofixation rates, been 7.5% (v/v) CO2 concentration, the one

that revealed the best result (1.34 g CO2 L-1

d-1). When compared to air-ambient condition, all cultures

(for this microalga), under higher CO2 concentrations increased their theoretical CO2 biofixation,

however it is not a linear increment, since a low value is achieved under the intermediate CO2 level -

5% (v/v).

Both C. vulgaris and C. protothecoides presented lower theoretical CO2 biofixation rates when

compared to S. obliquus, except for the culture of C. protothecoides under 10% (v/v) CO2

concentration. The results attained by C. vulgaris, despite of being the lowest of this study, are much

higher than the ones from Chinnasamy et al. (2009): for ambient (0.035%) and intermediate levels

(6% CO2), C. vulgaris ARC1 could fix 0.018 and 0.038 g CO2 L-1

d-1, respectively under 47 μmol m

-2 s

-1

photon density.

C. protothecoides stood out with the highest CO2 biofixation rate (1.98 g L-1 d-1), when 10%

(v/v) CO2-enriched air is imposed to the culture. In comparison with literature, this is an excellent

value. Several studies (for several species of microalgae) presents CO2 biofixation rates below 1 g L-1

d-1 when considered high percentages of CO2 concentrations (Ho et al., 2010; de Morais and Costa,

2007; Huntley and Redalje, 2006; Voltolina et al., 2005; Yun et al., 1997). Since these results are

based on maximum productivities in both cases, S. obliquus and C. protothecoides seem to be

prominent candidates to be applied in CO2 mitigation systems with relatively high CO2 concentration

added to the cultures.

5.2 Influence of CO2 concentration on microalgae biomass quality

5.2.1 Oil content

Lipids are the most desirable component for biodiesel production (Mata et al., 2010; Gouveia

and Oliveira, 2009). Thus, it is important to evaluate the oil content (% wt.) for each microalgae strain

under the different CO2 concentrations tested. Final oil content is presented in Figure 5.5.

% CO2 (v/v)

concentration

Theoretical CO2 biofixation rate (g CO2 L-1

d-1

)

S.obliquus C. vulgaris C. protothecoides

0.035% (Air) 0.36 0.48 0.47

2.5% 1.14 - -

5.0% 0.89 0.74 0.22

7.5% 1.34 - -

10% 1.21 0.48 1.98

15% 1.19 - -

39

In a general way, S. obliquus presented the higher oil content, however without a logical. In

this study, the higher values were obtained for 2.5% and 15% CO2 tested concentrations, with 26.3%

and 25.3% weight of dry biomass, respectively. According to Tang et al. (2011) it was expected that

the total oil content of S. obliquus, showed raising trends with the increase of CO2 concentration.

The 2.5% (v/v) CO2 concentration coincide to the one where the average productivity was

superior (Figure 5.4). This was an unexpected result as normally the higher lipid synthesis occur when

the productivity is low, due to a (some) stress(es) factor(s) (Gouveia et al. 2009; Gouveia and Oliveira,

2009). In the literature is reported that under concentrations proximate of the 2.5% tested CO2

concentration, oil content presents values of 8.2% (against 26.3% in this study). For CO2

concentrations close to 15% CO2 levels, higher results were achieved, around 33% of oil content

(25.3% in this work) (Baky et al., 2012).

C. protothecoides showed the highest lipid content under 10% CO2-enriched air. Accordingly

to Miao and Wu (2004), the lipid content reached for this specie autotrophically, was 14.6%, less 3.8%

that was achieved in this study. C. vulgaris was the microalga that presented the worst result in terms

of lipid content. Previous studies shown that this specie can reach oils between 20 to 50%, which

would make C. vulgaris a good candidate for biodiesel production (Bhola et al., 2011; Lv et al., 2010;

Liu and Wang, 2008; Illman et al., 2000). However in this study, the lipid content does not overcome

10% in all the CO2 concentration tested. Possibly, the specific environmental conditions of this

experience were not the most satisfactory for lipid accumulation. However use of high concentration of

CO2 could be a very interesting strategy to increase the growth rate and biomass productivity,

Figure 5.5 - Oil content in % (mass of oil per mass of dried biomass) from the cultures of S.


s-1

, 1vvm),

subjected to the different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v)



40

especially when a flue gas from a pollutant industry could be used (Lam and Lee, 2013), with evident

environmental benefits.

5.2.2 Fatty acid profile

In addition to establish the oil content from microalgae cultures, it is equally important to obtain

an appropriate composition of fatty acid profiles, as they have a strong effect on the quality of the

biodiesel produced. Among the unsaturated fatty acids, special attention should be taken in relation to

the α-linolenic methyl ester (C18:3 and polyunsaturated methyl esters (≥4 double bonds content due

to the EN 14214 (2003) that specifies limits of 12% and 1%, respectively.

The fatty acid profile was determined for the three microalgae species, for all CO2

concentrations tested by GC-FID. The chromatographs represents the profile of each microalga,

where each peak corresponds to a specific fatty acid composing the respective oil (eg., Figure 5.6 for

C. protothecoides).

Table 5.2 depicted the fatty acid profile of S. obliquus for the different CO2 concentrations

tested. Gas chromatography revealed, in a general way, that C16:0 (palmitic acid), C18:1 (oleic acid)

and C18:2 (linoleic acid) were the main known fatty acids present in the S. obliquus. Prior studies

shown that high levels of CO2 enhance the production of polyunsaturated fatty acids such as C18:2

and C18:3 (Tang et al., 2011; Ota et al., 2009).

Figure 5.6 - Fatty acid profile from the oil of one of the duplicates of C.

protothecoides [5% (v/v) CO2 concentration, ≈ 28º C; 74 µmol photons m-2

s-1

; 1vvm],

obtained by GC.

41

Table 5.2 - Fatty acid profile for the different CO2 concentration (v/v) tested for S. obliquus and respective

standard deviation among the average of two duplicates.

Among the fatty acid profile it is not shown a significant changes neither a perceptible trends

on the increase or decrease of a particular fatty acid under the different tested CO2 concentrations. α-

Linoleic acid (C18:3), for all CO2 concentrations, assumed values under 12% and do not presented

polyunsaturated methyl esters (≥4 double bonds , what makes this specie adequate to produce

biodiesel with good quality. Moreover, the unsaturated fatty acids were the main components of the

total fatty acids for S. obliquus which suggested that the biodiesel produced from this microalga would

have low viscosity. The highest amount of unsaturated fatty acids was obtained under 5% CO2

(63.8%). The unsaturated, especially polyunsaturated fatty esters have lower melting points, which

were desirable for the improvement of the low-temperature properties of biodiesel, taking into account

that C18:3 and polyunsaturated methyl esters (≥4 double bonds should be under the values

stipulated by the EN 14214 (2003) (Tang et al., 2011).

Table 5.3 presents the fatty acid profile of C. vulgaris, for the different CO2 concentrations

tested. The known fatty acids methyl ester composition was mainly C16:0, C18:1 and C18:3.

S. obliquus

Fatty Acid Air 2.5% CO2 5% CO2 7.5% CO2 10% CO2 15% CO2

C12:0 - - - - - - C14:0 0.5±0.1 0.3±0.0 0.3±0.1 0.3±0.1 0.4±0.1 0.4±0.0 C16:0 24.7±1.3 24.9±0.1 23.6±0.1 22.0±0.4 24.3±1.4 25.8±0.1

C16:1 1.4±0.1 1.4±0.1 1.3±0.3 1.9±0.0 1.5±0.3 1.1±0.0 C18:0 2.2±0.4 4.4±0.1 3.4±0.2 3.3±0.1 2.8±0.4 3.3±0.0 C18:1 42.8±3.8 40.1±0.3 42.8±1.5 42.0±0.5 41.6±2.6 41.2±0.2

C18:2 9.9±1.3 12.6±0.1 10.3±0.2 10.3±0.0 9.0±1.4 9.1±0.1

C18:3 8.7±3.1 7.1±0.2 9.4±1.0 9.3±0.1 9.7±2.4 7.9±0.1 C20:0 0.2±0.1 0.6±0.0 0.2±0.0 0.2±0.0 0.2±0.1 0.7±0.0 C20:1 0.1±0.0 - - - - - C22:0 0.1±0.0 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.0 C22:1 - 0.2±0.1 - - - 0.8±0.0 C24:0 0.1±0.0 - - - - - Other 9.3±1.3 8.2±0.2 8.5±0.1 10.5±0.7 10.3±0.9 9.5±0.1

42

Table 5.3 - Fatty acid profile for the different CO2 concentration (v/v) tested for C. vulgaris, with respective

standard deviation among the average of two duplicates.

It is clearly observed that under high CO2 concentrations the saturated fatty acid such as

C16:0 tents to decrease, as well as C18:1, in opposition of C18:3 which the tendency is to increase.

This can occur as the increase in CO2 concentration could lead to a relative decrease in O2

concentration that might affect the enzymatic desaturation (Vargas et al., 1998), increasing the

polyunsaturated fatty acid content. On the other hand, low CO2 concentrations would favor the

accumulation of saturated fatty acids (Ota et al., 2009; Tang et al., 2011). In addition, it was found that

oleic fatty acids (C18:1) tents to decrease with the increase of CO2, whereas linoleic fatty acids

(C18:2) content do not present perceptible trends when higher CO2 supply is applied. Along the CO2

tested concentrations, C. vulgaris biomass despite of has no polyunsaturated methyl esters (≥4 double

bonds), the α-linolenic acid presented a value of 22.0% (air), 30.5% (5% CO2) and 28.3% (10% CO2),

overcoming the 12% limit specified by EN 14214, which is impeditive for a good quality biodiesel.

Even so, the oil can be properly mixed with other oils or be used as raw material for other biofuel

production processes (Gouveia and Oliveira, 2009). Additionally, the unsaturated fatty acids do not

overpass the 54% for all tested CO2 concentrations, a value far short compared to the one presented

for S. obliquus. It is suggested that improved low-temperatures properties for biodiesel could be

impaired.

C. vulgaris

Fatty Acid

Air 5% CO2 10% CO2

C12:0 - - - C14:0 0.7±0.3 2.2±0.2 0.8±0.3 C16:0 23.7±7.3 21.6±0.3 16.5±6.4

C16:1 1.0±0.9 1.8±0.1 1.8±0.3 C18:0 1.8±1.5 0.6±0.0 1.0±0.3 C18:1 21.3±13.9 12.5±0.4 13.7±0.3

C18:2 9.2±0.5 7.5±0.3 10.4±0.3 C18:3 22.0±9.8 30.5±0.0 28.3±0.7

C20:0 - - - C20:1 - 0.4±0.0 - C22:0 - - - C22:1 - C24:0 - - - Other 20.3±13.5 22.9±0.4 27.5±3.6

43

Table 5.4 presents the fatty acid profile of C. protothecoides, for the different CO2

concentrations tested.

Table 5.4. - Fatty acid profile for the different CO2 concentration (v/v) tested for C. protothecoides with the

respective standard deviation among the average of two duplicates.

Like C. vulgaris, C. protothecoides biomass presented C16:0, C18:1 and C18:3, as the major

known fatty acids in its oil composition. However, it seemed to respond in a different way, when CO2

levels rise. Instead of a decrease, it was verified an increase on the saturated fatty acid, palmitic

methyl ester (C16:0). Moreover, the α-linolenic methyl ester (C18:3) seemed to have an increment

when are considered the 5% CO2 levels and a decay on its composition is registered, when CO2

concentration increase. This different fatty acid composition, along the CO2 concentrations tested,

could be associated to a variable performance of desaturase enzyme in specific for this strain of

microalga. A similar behavior was reported in Chlorella kessleri, where low CO2-levels promote

desaturation activity of pre-existing fatty acids (Sato et al., 2003). Concerning the differing results, it

seems to reinforce the idea that lipid composition is strain-dependent, regarding the CO2 influence on

microalgae cells.

Polyunsaturated methyl esters (≥4 double bonds are not present, but the α-linolenic acid

along the CO2 concentrations tested present a value of 23.9% (air), 30.9% (5% CO2) and 18.8% (10%

CO2), overcoming the 12% limit specified by EN 14214, which is, again, not permitted to produce a

good biodiesel. Nevertheless, the same statement could be employed as for C. vulgaris, i.e. the oil

produced by C. protothecoides could be used if properly added and mixed with other oils. Regarding

the percentage of unsaturated fatty acids, in opposition to C. vulgaris, it was much higher, namely for

air (56.7%) and for 10% CO2 (60.2%). The results indicate a good biodiesel quality in terms of

viscosity, as it was stated for S. obliquus.

The different fatty acid profiles among the three microalgae are clearly strain-dependent.

C. protothecoides

Fatty Acid

Air 5% CO2 10% CO2

C12:0 - 0.2±0.0 - C14:0 0.8±0.7 2.3±0.1 0.6±0.1 C16:0 18.7±1.0 22.0±0.6 23.9±0.9

C16:1 1.0±1.0 1.7±0.1 1.7±0.0 C18:0 1.0±0.1 0.8±0.1 1.5±0.0 C18:1 18.0±5.0 11.9±0.8 27.0±1.2

C18:2 13.8±2.3 7.7±1.0 12.3±0.6 C18:3 23.9±1.2 30.9±2.2 18.8±1.1

C20:0 0.2±0.3 - 0.1±0.0 C20:1 - 0.3±0.0 0.4±0.0 C22:0 2.2±2.8 - - C24:0 - - - Other 20.4±0.9 22.2±0.1 13.7±1.4

44

5.2.3 Total pigment content and profile

5.2.3.1 Total chlorophyll content

To make an overall analysis on the influence of CO2 on value-added products for an

economical valorization, it was performed a more detailed study on quantification and profile on

pigments, at the end of each microalgae culture. In these regards, the quantification of total chlorophyll

content (mg L1), including chlorophyll a and b, was evaluated as it is shown in Figure 5.7.

Regarding this results it is important to note the lack of some standard deviations (Figure 5.7).

Some of the duplicate were lost during the material handling (broken test tubes during centrifugation),

and unfortunately it was not possible to repeat due to lack of the respective samples.

It can be observed that S. obliquus have the lowest content on chlorophyll than both Chlorella.

The concentration ranged between 1.4 to 3.5 mg L-1

. This microalga, generally do not presented great

amounts of chlorophyll, despite of having the best specific growth rates (Figure 5.3) (which represents

a good growth performance). Actually, not always the increase on biomass reflects population growth

as in certain conditions it can represent an increase of storage compounds (Madigan et al., 2010).

This is corroborated by the results obtained from the oil content (Figure 5.5), where S. obliquus

presented higher values than both Chlorella. It seems that S. obliquus metabolism is more targeted to

fix carbon in form of storage compounds, in this case lipids, with a relatively high composition, than

pigments.

Thereafter, C. vulgaris presented values of chlorophyll content, ranging between 5.1 to 8.4 mg

L-1

, along the CO2 tested concentrations. It was verified a marked increasing trend on the total

chlorophyll content, when CO2 concentrations become higher. Apparently, this strain was more

Figure 5.7 – Representation on the total chlorophyll content (mg L-1

) on the extracts, from

the cultures of S. obliquus, C. vulgaris and C. protothecoides (≈ 28ºC; 74 µmol photons m-2

s-1

; 1vvm), subjected to different CO2 concentrations tested: Air ( ); 2.5% (v/v) CO2 ( );

5% (v/v) CO2 ( ); 7.5% (v/v) CO2 ( ); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical

bars represent standard deviations among the average of two duplicates.

45

capable to use carbon, for pigment accumulation instead production of storage compounds, such as

lipids, in the tested conditions. In fact, the chlorophyll content of C. vulgaris is the one of the most

elevated on nature (Seyfabadi et al., 2011).

C. protothecoides attained the highest concentrations with emphasis to the control culture.

The total chlorophyll content ranged between 1.2-13 mg L-1

. The specific growth rate (Figure 5.3)

were not in accordance with the observed results on the total chlorophyll content, except for 5% (v/v)

CO2. For this concentration C. protothecoides presented a low chlorophyll content associated to a low

specific growth rate. These results reinforce the idea that something influenced negatively the growth

of this microalga, in the referred condition. Curiously, Miao and Wu, (2004) reported a value of 15 mg

L-1

of total chlorophyll content for C. protothecoides grown autotrophically with air, which is not far from

the result of the present study.

5.2.3.2 Pigment content

Thin layer chromatography (TLC) was used to identify the pigments present in the sample in

the acetone extracts of the cultures. Chlorophyll a, beta-carotene and lutein were the main pigments

detected.

In order to quantify the pigments on the extracts, namely the beta-carotene and lutein, it was

performed an HPLC analysis. The results are shown on Figure 5.8A and B.

It is highlighted the difference between scales on the graphs. This note reveals an important

dissimilarity on the content of each pigment evaluated in the different extracts. Regarding beta-

carotene, it was not reported, so far, an influence of CO2 on its accumulation. In this study, beta-

carotene stood out with low values, ranging between 0.04 and 0.14 mg L-1

(Figure 5.8A). The highest

Figure 5.8 – Representation of the content (mg L-1

) on beta-carotene (A) and lutein (B)

pigments, on the extracts from the cultures of S. obliquus, C. vulgaris and C.


s-1

; 1vvm), subjected to different CO2

concentrations tested: Air ( ); 2.5% (v/v) CO2 ( ); 5% (v/v) CO2 ( ); 7.5% (v/v) CO2 (

); 10% (v/v) CO2 ( ); 15% (v/v) CO2 ( ). Vertical bars represent standard deviations

among the average of two duplicates.

46

value belongs to C. protothecoides (10% CO2), yet very low. It is possible to observe a slightly

increase on both Chlorella, with the increase on CO2 concentration. The low results were expected for

all extract samples, since the microalgae cultures were not subjected to stress during the growth. It is

well documented that high salinity, stress temperature, light intensity and nitrogen limitation favor beta-

carotene accumulation (Campenni’ et al., 2013; Lv et al., 2010; Gouveia, 1996). An example could be

done by Dunaliella salina which under stress conditions could accumulate beta-carotene up to 12% of

the algal dry weight. It should be noted that even without imposed stresses, Dunaliella salina is one of

the few microalgae that have a high content of beta-carotene (Del Campo et al., 2007).

Figure 5.8B exhibits the results for the lutein content on the extracts, from the cultures

subjected to different CO2 concentrations. The concentration on lutein ranged between 0.01 and 5.3

mg L-1

. The influence of CO2 in the accumulation of lutein seemed to be more relevant in comparison

with beta-carotene. For both S. obliquus and C. vulgaris, for all tested CO2 concentrations barely

overcame 1 mg L-1

with a few exceptions, but with no relevance. C. protothecoides obtained the lowest

and the highest results. In this strain, supplement on the CO2 seemed to have some positive influence

on the lutein content. However the control culture presented the best value in this context. The

discrepancy for the 5% (v/v) CO2 levels result depicts the low performance on growth for this strain.

Values from the literature, range from 3.4 to 4.6 mg per gram of dry weight for different microalgal

strains and experimental conditions ( Del Campo et al., 2007; Campenni' et al., 2013). 4.1 mg g-1

of

dry weigh is the best value obtained in autotrophic growth at laboratory scale for C. protothecoides

(Campenni’ et al., 2013). Attending that DCW, in the control (air) for C. protothecoides, in the last day

of the culture, was an average of 1.83 g L-1

(data not shown), the value of lutein would be nearly 2.90

mg g-1

of dry weight, 1.4 times less than the cited value.

5.3 Influence of CO2 aeration rates on microalgae growth

In order to understand the influence of the aeration rate on microalgae growth, a study under

different flow rates, at 5% (v/v) CO2 concentration was performed. S. obliquus was the chosen

microalga cultivated over 15 days on batch mode using Bristol’s Medium, at approximately 28ºC and

under 74 µmol m-2

s-1

of light intensity. The tested CO2 flow rates were: 0.25; 0.50; 0.75 and 1 vvm.

pH was monitored for the different cultures and ranged between 6.11 and 7.37.

It is important to note that the initial study encompassed only air [0.035% (v/v)], 5% (v/v) and

10% (v/v) CO2 concentrations. S. obliquus had the best results in terms of specific growth rate [under

5% (v/v) CO2], at the time. This is why this microalga and this specific CO2 concentration were chosen

for the CO2 aeration rate study.

To observe the effect of the different CO2 flow rates on S. obliquus growth the parameters

used were the previous ones, such as maximum dry cell weight (DCWmax), specific grow rate (µ) and

average productivity (Pa). Moreover, a more insightful study was performed in terms of CO2 mitigation

such as the theoretical and the real biofixation of CO2 were estimated and compared.

47

Regarding the DCWmax, presented on Figure 5.9A, values ranged between 2.6 and 3.0 g L-1

,

without significant differences among them. However the best value was attained on the culture

aerated with 0.75 vvm (3.0 g L-1

).

It is possible to conclude that there is no remarkable influence on the DCWmax, when different

CO2 flow rates are applied. Such behavior could be explained by mild conditions in terms of CO2

concentration and aeration rates used in this study. In the literature, different results are depicted. For

low flow rates (0.003 vvm) cultures of S. obliquus CNW-N, it were reported DCWmax approximately of

1.9 g L-1

(Ho et al., 2010). Other values are reported for S. obliquus, 0.3 vvm aeration rates [for

intermediate 6% (v/v) CO2 levels], it were registered DCWmax equals to 1.56 g L-1

, (de Morais and

Costa, 2007). Whilst C. vulgaris cultures, bubbled with 0.1, 0.4 and 0.7 airstreams [6% (v/v) CO2

concentrations], reported values of 6.8, 10 and 8.9 g L-1

, respectively (Anjos et al., 2013). Attending to

the reported values (Ho et al., 2010; de Morais and Costa, 2007), and despite of CO2 concentration

can vary, generally the obtained results showed to be higher in this study. On the other hand,

compared to C. vulgaris (Anjos et al., 2013), it is possible to observe a marked increasing trend of the

DCWmax with the flow rate increase, which was not perceptible in this study. Again, the influence of

aeration rates could also be strain-dependent.

Considering the specific growth rate (µ), represented on Figure 5.9B, S. obliquus cultures

under different CO2 aeration rates, exhibited values ranging between 0.49 and 0.76 d-1

, The

Figure 5.9 – Representation of the biomass performance of S. obliquus [5% (v/v) CO2 ; ≈ 28º C; 74

µmol photons m-2

s-1

], under different CO2 flow rates: 0.25 vvm ( ); 0.50 vvm ( ); 0.75 vvm ( ); 1

vvm ( ) in terms of (A) Maximum dry cell weight; (B) Specific growth rate; (C) Average

productivity. Vertical bars represent standard deviations among the average of two duplicates.

48

immediate observation are the lowest values, presented by the intermediate tested flow rates (0.50

and 0.75 vvm), with 0.55 and 0.49 d-1

, respectively. The results seem to be ambiguous, since the

highest µ belongs to the opposite aeration rates 0.25 vvm (µ = 0.66 d-1

), and 1 vvm (µ = 0.76 d-1

).

One possible explanation is that the higher turbulent motion of liquid intensifies the movement

of cells, at the region adjacent to the photobioreactor wall, leading to an enhanced use of light by

microalgae, which clears the better grow performance of the S. obliquus culture, under 1 vvm flow

rate. On the other hand, when low flow rates occur in the culture, the gas bubble retention time is

higher and the gas mixture dissipation for the environment (to the outside of the photobioreactor) is

attenuated, which implies an efficient utilization of CO2 by microalgal cells (Ryu et al., 2009) and

consequently better growth performance. More detailed investigations are needed to further validate

these contradictory results.

Towards average productivity (Pa), observed in Figure 5.9C, values ranged between 0.30 and

0.40 g L-1

d-1

. It was noticed a slightly productivity increase, when are considered the aeration rates of

0.75 vvm (Pa = 0.39 g L-1

d-1

) and 1 vvm (Pa = 0.37 g L-1

d-1

), compared to the lower ones. Like it was

explained previously, when a good mixing/agitation of liquid culture is provided, productivities are likely

to increase, since it is prevented cell sedimentation and light exposure is equally ensured (Gouveia,

2011; Ryu et al., 2009).

5.3.1 CO2 biofixation and mitigation

In order to analyze the real mitigation by S. obliquus microalga, subjected to different flow

rates, theoretical and real CO2 biofixation were assessed and compared. Figure 5.10 shows the

compared results. The immediate observation is that real CO2 biofixation seems to be higher than the

estimated CO2 biofixation, based on the maximum productivity. Actually, values for theoretical

biofixation ranged between 0.78 and 1.20 gCO2 L-1

d-1

, whereas real CO2 biofixation ranged between

1.65 and 2.55 gCO2 L-1

d-1

. Simply increasing on CO2 aeration rates from 0.25 to 1 vvm in S. obliquus

culture medium [5% (v/v) CO2 concentration], apparently resulted in an increase on the real mitigation.

In fact this is more pronounced attending to the highest CO2 aeration rate tested (1 vvm). These

results could be explained by the increased CO2-gas mixture in the medium during the culture growth

and are supported by the higher specific growth rate (Figure 5.9B) achieved for 1 vvm condition.

Besides, CO2 harnessing could favor more biomass concentration as a result of higher culture density,

and consequently enhance CO2 mitigation. However not always CO2 aeration rate is synonymous of

improved CO2 biofixation rate or CO2 removal efficiency. Literature is far from consistent across this

aspect. Li et al. (2011) showed that an increasing flow rates from 0.1 to 0.5 vvm in S. obliquus WUST4

culture, culminated in a decreasing of CO2 removal efficiency from 67 to 20% (Li et al., 2011). Anjos et

al. (2013) pointed out intermediate flow rates (i.e. 0.50 vvm), combined with intermediate CO2

concentrations [6.5% (v/v) CO2], favors good result in terms of CO2 biofixation rate efficiency (2.2 g

CO2 L-1

d-1

). Furthermore and contradicting the reported data, the CO2 biofixation increment is

49

registered, when 0.25 to 0.50 flow rates are considered, under 5% (v/v) CO2 concentration, for

cultures of Chlorella sp. MT-7 and sp. MT-15 (Ong et al., 2010).

The contradictory results might arise as a consequence of various culturing parameters

(biomass concentration, nutrients, light regime and types of PBRs), and how individual microalga

specie behave and affect each culture system.

5.4 Influence of CO2 aeration rates on microalgae biomass quality

5.4.1 Oil content

The effect of the imposed CO2 aeration rates on S. obliquus cultures [5% (v/v) CO2

concentration], was investigated on the final oil content (% wt.). Figure 5.11 presents the results and

showed an apparently moderate rising tendency when CO2 flow rates increase. The lowest value was

achieved by the S. obliquus culture subjected to 0.25 vvm CO2 flow rate (20.1% wt.) and the better

result was attained under the 1vvm of CO2 flow rate (28.4% wt.). Apparently, higher CO2 aeration

favours the accumulation of lipids. Such behaviour was already demonstrated for C. vulgaris, but not

for a wide window of CO2 flow rates values, like in this study. Zheng et al. (2012), under the same 5%

CO2 levels during culture growth, demonstrated that C. vulgaris oil content increased when the CO2

aeration rate was increased from 0.1 to 0.5 vvm). However, a further increase in CO2 aeration rate

discouraged the lipid accumulation of the strain (Zheng et al., 2012).

Figure 5.10 – Comparison between the theoretical CO2 biofixation ( ) and the real CO2 biofixation

( ), along the different aeration rates tested, in S. obliquus [5% (v/v) CO2 concentration; ≈ 28ºC; 74

µmol photons m-2

s-1

]. Vertical bars represent standard deviations among the average of two

duplicates.

50

5.4.2 Fatty acid profile

The composition of fatty acids in the S. obliquus cultures [5% CO2 concentration], for all flow

rates tested, was investigated. The analysis was performed by GC and the data is presented on Table

5.5.

Table 5.5 - Fatty acid profile for the different aeration rates tested for S. obliquus [5% (v/v) CO2 concentration; ≈

28ºC; 74 µmol photons m-2

s-1

] and respective standard deviation among the average of two duplicates.

It is observed that C16:0 (palmitic acid), C18:1 (oleic acid) and C18:2 (linoleic acid) were the

main known fatty acids present for all flow rates tested in the S. obliquus. In fact this was already

showed in the Section 5.2.2. Palmitic methyl ester (C16:0) was very constant with the increase of

aeration rates, however suffered a slightly decrease 1 vvm. A similar behavior was denoted by Zheng

et al. (2012) for C. vulgaris, under 5% CO2 concentration, when CO2 flow rates increased. Regarding

Fatty Acid

Aeration rates

0.25 vvm 0.50 vvm 0.75 vvm 1 vvm

C12:0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 C14:0 0.3±0.0 0.3±0.0 0.3±0.1 0.3±0.0 C16:0 24.6±0.9 24.7±1.7 24.7±1.1 23.6±0.2

C16:1 1.1±0.1 1.0±0.1 1.1±0.1 1.1±0.1 C18:0 3.8±0.1 3.9±0.2 3.7±0.0 3.4±0.1 C18:1 44.2±2.0 46.3±3.7 44.7±2.4 45.7±2.5

C18:2 9.4±2.5 9.5±2.0 9.2±2.3 8.7±2.5

C18:3 6.4±2.5 6.3±2.9 7.3±1.7 7.9±1.2 C20:0 0.2±0.0 0.3±0.1 0.2±0.0 0.2±0.1 C20:1 - - - - C22:0 0.2±0.0 0.2±0.0 0.2±0.0 0.2±0.1 C22:1 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 C24:0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 Other 9.8±1.9 7.5±0.7 8.6±0.4 8.9±0.7

Figure 5.11 - Oil content in % (mass of oil per mass of dried biomass) from the cultures of S.

obliquus [5% CO2 (v/v) concentration; ≈ 28ºC; 74 µmol photons m-2

s-1

], subjected to different CO2

flow rates: 0.25 vvm ( ); 0.50 vvm ( ); 0.75 vvm ( ); 1 vvm ( ). Vertical bars represent standard

deviations among the average of two duplicates.

51

the unsaturated fatty acids, such as C18:1, apparently there is an irregular fluctuation along the tested

aeration rates, do not presenting discernible trends of increasing or decreasing, on a particular

direction. However linoleic acid (C18:2) seemed to have a slightly drop on its values, when CO2 flow

rates increase, but nothing substantial. In addition, the percentage of unsaturated fatty acids was

relatively high: above 60% for all tested flow rates. The best value achieved was 63.4%, under 1 vvm

flow rate; a good indicator for desirable low-temperature properties of biodiesel.

Concerning EN 14214 (2003), the α-Linoleic acid (C18:3), assumed values under 12% and do

not presented polyunsaturated methyl esters (≥4 double bonds , what makes this specie adequate to

generate biodiesel with fine quality, regardless the tested CO2 aeration rates.

52

6. Conclusions and Future work

The aim of the present work was to determine the best microalga specie (among

Scenedesmus obliquus, Chlorella protothecoides and Chlorella vulgaris) to mitigate CO2. The cultures

were subjected to different CO2 concentrations and aeration rates (for S. obliquus) and their influence

on biomass growth, average productivity and theoretical CO2 biofixation was evaluated. Thereafter,

the influence on biomass quality (oil content and fatty acid profile, and pigment content and profile)

was also evaluated. The final purpose was to verify the potential for ultimate economic valorization

after removing the CO2 and produce a biomass with quality to biodiesel production and pigment use.

In a general way, S. obliquus and C. protothecoides seem to be good potential candidates to

CO2 bio-mitigation systems conjugated with the production of value added-products.

S. obliquus demonstrated to be the best microalga in terms of growth (µ = 0.99 d-1

), under

7.5% (v/v) CO2 concentration and average productivity (Pa = 0.37 g L-1

d-1

), under 2.5% (v/v) CO2. The

study on different CO2 aeration rates (0.25; 0.50; 0.75 and 1.0 vvm), for this microalga, presented

ambiguous results in terms of growth, which would require a more detailed investigation. Low flow

rates associated to good growth performances would be more desirable, in order to save costs on

mitigation process.

Since productivity is economically important for the overall microalgal CO2 biofixation process,

not only due to attaining more microalgal biomass for commercial uses but also for shortening the

cultivation time, it is suggested that S. obliquus is the most appropriate specie in these regards.

Moreover, S. obliquus seems to be more targeted to produce oils for biodiesel production, since

achieved the best oil content at 2.5% and 15% (v/v) CO2 levels with 26.3% and 25.3%, respectively.

In addition, only S. obliquus specie evidenced a good profile composition (in all tested CO2

concentrations and flow rates) for suitable direct biodiesel production, according to the EN 14214.

C. protothecoides conjugated a good grow performance (best DCWmax = 5.8 g L-1

) at higher

CO2 levels with the higher theoretical CO2 biofixation rate (1.98 g CO2 L-1

d-1

) both under 10% (v/v),

suggesting a good prospective for biomass production and CO2 mitigation. Additionally, this microalga

evidenced a high potential to produce value-added compounds, especially chlorophyll (13 mg L-1

) and

lutein (5.3 mg L-1

). C. protothecoides could be a good potential candidate to mitigate CO2, producing

good amounts of biomass, and contributing to commercial valorization applied to several industries,

such as food/feed and/ or bio-energy production or even for pharmaceutical.

For all tested microalgae, increment on CO2 did not favor beta-carotene accumulation.

However, in a CO2-mitigation system, biomass recovery and value-added products, such as lutein,

would be much more prominent for application in range of commercial areas.

Regarding this study, more detailed investigation should to be done namely on C. vulgaris and

C. protothecoides growth. Experiments with 5% (v/v) CO2 should be repeated, in order to understand

why the growth performance was not good, since the major literature contradicts these results. The

CO2 aeration rates should also be investigated along with CO2 concentrations present to better

53

understand their influence on microalgae growth. Finally, a more detailed study on real CO2 mitigation

would be desirable.

Possible guidelines for future work would be more basic research on microalgae strains that

can potentially bio-mitigate CO2, cultivation strategies to increase CO2 fixation, tendencies of CO2

fixation/ high added-value product synthesis and taking full advantage of the formed products (oils,

pigments, antioxidants, aminoacids and others) that will increment the economic feasibility of the

whole process.

54

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Abreu, A. P., Fernandes, B., Vicente, A. A., Teixeira, J., & Dragone, G. (2012). Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source. Bioresource Technology, 118, 61–6. doi:10.1016/j.biortech.2012.05.055

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Annex I – CO2 Calibration curve

The CO2 calibration was made to adjust the real CO2 concentration on the entrance to the

photobioreactors, in order to obtain the specific CO2 concentrations for the experiments. The

calibration was performed using four standards with 1,3, 5 and 20% of CO2.

It was obtained the line equation:

% real CO2 = 0.925 × % measured CO2 – 0.3701

The correlation coefficient R2 obtained was equal to 0.9994.

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Annex II – Beta-carotene calibration curve

Beta-carotene calibration curve was used to quantify this pigment. The calibration curve was

obtained, using the corresponding peak areas on HPLC versus know concentrations of beta-carotene.

The peaks of beta-carotene were obtained at 450 nm of wavelength.


Peaks area = 253.89 × beta-carotene concentration – 1.7166

The correlation coefficient R2 obtained was equal to 1.

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Annex III – Lutein calibration curve

Lutein calibration curve was used to quantify this pigment. The calibration curve was obtained,

using the corresponding peak areas on HPLC versus know concentrations of lutein.

The peaks of lutein were obtained at 450 nm of wavelength.


Peaks area = 139.67 × lutein concentration – 0.4386

The correlation coefficient R2 obtained was equal to 0.9998.

influence of co on scenedesmus obliquus chlorella vulgaris and€¦ · oil, pigment content and...

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