Western UniversityScholarship@Western Electronic Thesis and Dissertation Repository February 2013 Batch and Continuous Studies of Chlorella Vulgaris in Photo-Bioreactors Claudia Sacasa Castellanos The University of Western Ontario Supervisor Amarjeet Bassi The University of Western Ontario Follow this and additional works at: http://ir.lib.uwo.ca/etd Part of the Biochemical and Biomolecular Engineering Commons Recommended Citation Sacasa Castellanos, Claudia, "Batch and Continuous Studies of Chlorella Vulgaris in Photo-Bioreactors" (2013). Electronic Thesis and Dissertation Repository. Paper 1113. This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact
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BATCH AND CONTINUOUS STUDIES OF Chlorella vulgaris IN PHOTOBIOREACTORS (Spine title: Batch & Continuous Study of C. vulgaris in Photo-bioreactors) (Thesis format: Monograph) by Claudia Sacasa Castellanos Graduate Program in Chemical and Biochemical Engineering A thesis submitted in partial fulfillment of the requirements for the degree of M.E.Sc. in Chemical and Biochemical Engineering The School of Graduate and Postdoctoral Studies The University of Western Ontario London, Ontario, Canada January 2013 © Claudia Sacasa Castellanos 2013 THE UNIVERSITY OF WESTERN ONTARIO School of Graduate and Postdoctoral Studies CERTIFICATE OF EXAMINATION Supervisor Examiners ______________________________ Dr. Amarjeet Bassi ______________________________ Dr. Ernest Yanful Supervisory Committee ______________________________ Dr. Shahzad Barghi ______________________________ Dr. Mita Ray ______________________________ Dr. Dimitre Karamanev The thesis by Claudia Sacasa Castellanos entitled: Batch and Continuous Studies of Chlorella vulgaris in Photo-bioreactors is accepted in partial fulfillment of the requirements for the degree of M.E.Sc. in Chemical and Biochemical Engineering ______________________ Date _______________________________ Chair of the Thesis Examination Board ii Abstract Microalgae are unicellular, photosynthetic microorganisms, they have minimal nutrient requirements and are being used as source materials for a variety of products such as protein rich nutritional supplements, pharmaceutical chemicals and pigments (used in food and cosmetics). They also grow extremely fast, in comparison to plants and many microalgae strains are exceedingly rich in oil which makes them great candidates for the production of biodiesel. The strain Chlorella vulgaris, has shown a great promise as a source of oil and as being able to help in the treatment of wastewater. Despite all the advantages , the high cost of production in bioreactors still remains a limitation and much research is required. The production of microalgae (Chlorella vulgaris) in different photobioreactor configurations (4L flask, tubular and stirred tank reactor) was investigated, using different media formulations and in both batch and continuous operation (for PBR and CSTR). The effect of dilution rate and mixing speed were both studied in the case of CSTR. The highest overall specific growth rate (µ) was found when growing C. vulgaris in continuous operation in a tubular photo-bioreactor, µ = 0.7 d-1, which also reached the maximum biomass concentration of 0.79 g/L. Keywords Microalgae, Chlorella vulgaris, tubular photo-bioreactor, stirred tank reactor, continuous operation iii to Jésus Moreira and Carolyn Moore for always being there for me and most importantly to my little sister Magaly who has been and will continue to be the main motivation for me to do better. Amarjeet Bassi. Brian Dennis as I asked him question after question and for giving me access to material and equipment essential to my research. that made it possible for me to be successful in the pursue and finalization of this project. who was my co-supervisor for part of my research. iv . Ajay Ray. and of all the friends that are like family to me. financially and emotionally. thank you for being so friendly. and Joanna Blom for his guidance and great support. I would like to thank Dr. I would also like to thank the members of the labs I visited and worked in for helping me with my experiments and for providing intelligent insights that made my work easier.Acknowledgments My sincere gratefulness goes to my supervisor Dr. of my mom Maribel and my brother Carlos. Lastly but not least I am very grateful for the support of my family. I am thankful for his encouragement to keep going and to constantly learn new things. support and understanding. I am grateful for the patience of Mr. His help. ................................................................................................................................................ iii Acknowledgments ......................................................................................................................................................................................................................................... 2 1...............3 Microalgae Cultivation ...........................2 Current Uses of Microalgae ....................................................... xii Chapter 1 ................ viii List of Figures ...2. 3 2....1........................................................................................................................ 3 2............................................................................. 3 2................................................................................. xi Nomenclature .......................1 Microalgae Growth Kinetics .. 2 Chapter 2 ................3............................................................ 9 2....................................................................................................................................................................................................2...........................................................................................1 What are Microalgae ........................................2 Factors Affecting Microalgal Growth ....................Table of Contents CERTIFICATE OF EXAMINATION . 3 2 Literature Review .......2...1 Nutrient Composition of Media ................................................................ 8 2.............................................................................................................................2 Light ..............1 Introduction on Microalgae....................................................... ii Abstract .............3 pH ......... v List of Tables ........................................................................................................................................................................................ 7 2..............1............................................................................................................ 1 1.....1 Research Objectives .................... 3 2............................................... iv Table of Contents ............ 7 2....................2 Thesis Overview ....................................................... 8 2........................................................................................... 1 1 Introduction ................................... ix List of Appendices ................................................................................................................... 9 v ....................................................................................................... ............................6.............................6.....4...... 12 2.................................4........................2 Other Media Formulation ...................................... 23 3..................................2...................... 29 3......... 26 3.............................................7 Determination of Dry Weight and Biomass Concentration .......9 Nutrient Analysis ........3........................ 28 3..........................4.........................5 Sampling of Photo-bioreactor .......2........ 15 2.......................................................................................3 Continuously Stirred Tank (CSTR) Photo-bioreactor ..................................... 14 2............................................................................... 21 3........................................... 19 3.2...........................................................1 4 L Conical Flasks ..... 28 3.............. 28 3.............................. 22 3.4 Air Supply ......... 19 3......................................................1 Microalgae Strain . 28 3........................................... 19 3 Materials and Methods ...............................................................6 Analysis of Samples ...................... 19 3..............................................4 Continuously Stirred Tank Reactors .......................................................................................................................................................................................5 Batch and Continuous Cultivation ......................................3........................................................... 22 3....................2 Growth Media ....................... 17 2......................................................................................................3........................................5 Lighting ................................ 25 3.....3 Inoculum and Strain Maintenance . 22 3..................................................................................................................................................................................................................................4 Photo-bioreactor Set-up ........ 19 3.................4 Importance of This Study......1 Bold’s Basal Media .................2 Photo-bioreactor Design .......................4..............3............8 Determination of CO2 Concentration .............. 26 3..................................2 pH .............2 Tubular Photo-bioreactor .....................................4........................... 18 Chapter 3 ..................................3 Tubular Photo-bioreactors ........................... 30 vi ... 28 3......1 Cell Count ................................................................................................. ................ 31 4.................. 31 4 Results and Discussions ........................................ 46 4........................ 46 4......................................... 35 4.......................................... 51 5 Conclusions and Recommendations ................................... 39 4..9..........................................................2 Batch Cultivation in Bold’s Media ................................ 33 4...................................................................6.....1 Nitrates .................... 30 Chapter 4 ........ ...........................2...........................................................1 Future Work ...............................................3 Batch Cultivation in Stirred Tank Photo-bioreactor............................2 Effect of Dilution Rate on Cell Growth ............... 31 4................................................................................................. 44 4............................... 68 vii ....................................4 Batch Cultivation in Tubular and Continuously Stirred Tank Photo-bioreactors ...........................1 Effect of Agitation Speed on Cell Growth ......................................................5 Semi-Continuous Cultivation in a Tubular Photo-bioreactor ... 42 4............ 31 4....................................................................................................1 Batch Cultivation in C30 and PCS-2 Media ......................................................3.................................. 63 Curriculum Vitae ........................ 52 References ................ 51 5............6 CSTR................2 Batch Cultivation in Tubular PBR................................................................................................................................................................1 Modified Bold’s Media with NaHCO3 .......................9...............................................2............................................ 48 Chapter 5 ..................................................................................................................................................................................1.................................... 54 Appendices .. .......................1 Batch Cultivation in 4L Flasks .. 30 3............ 33 4..............2 Phosphates....6.................................................. ......................... ..... ........... 46 Table 4.6: Biomass concentration at different dilution rates in a CSTR ........................................................2: Summary of continuous and semi-continuous cultivation of microalgae............1: Modified Bold’s Basal Media (Andersen................... ........ ...2: Parameters and results of CSTR experiments...............................................1: Comparison of Chlorella vulgaris growth in Tubular PBR with Modified Bold’s Media ..................... 21 Table 4....5: Biomass concentration at different mixing speeds in a CSTR .............................3: Comparison of Batch Studies with Chlorella vulgaris on different photobioreactor configurations...........................4: Steady state biomass concentration at different dilution rates in a tubular photo-bioreactor ............. 2005)......... 50 viii ................................... 2008).................................2: Other Media Formulations (Perez-Garcia et al....... 20 Table 3.... 36 Table 4........................ 48 Table 4........1: Comparison of crop-dependent and algae biodiesel (Schenk et al................................................ 2011)................ 43 Table 4............................ 40 Table 4.......List of Tables Table 2.. 13 Table 3........................................... 6 Table 2.... .................4: CSTR Diagram........................1: Microscopic image of Chlorella vulgaris UTEX 2714 ........... 25 Figure 3........................ 3 Figure 2...2: Tubular Photo-bioreactor Diagram .. 4 Figure 2...... 32 Figure 4... 23 Figure 3......................... Light Set-up On Stirred Tank Photo-bioreactor .........2: Various commercial applications of microalgae .........5.............................6... ...............................................................5: Tubular photo-bioreactor diagram .............................. 15 Figure 2................. Light Set-Up on Tubular Photo-bioreactor....................List of Figures Figure 2......................5: Comparison of change in biomass concentration of experiments with (A) CO2 enriched and (B) no added CO2 in air....... 10 Figure 2.....................3: Diagram of Ring Sparger .......... 34 Figure 4......... 9 Figure 2............................................... vulgaris in 4L flasks.......... 24 Figure 3........ ...... 27 Figure 3................3: Carbon dioxide equilibrium in water (Becker.......................................................................1: 4L Flask Set-Up.4: Microalgae batch growth profile ......6: Effect of mixing speed on Chlorella vulgaris cultivated in a CSTR ........................ 27 Figure 4.......................... 41 ix ...................... 16 Figure 3.............2: Change of pH with the addition of NaHCO3 ........................... 37 Figure 4.......................................................................3: Chlorella vulgaris growth in C30 (A) and PCS-2 (B) media................................ .............. 1994) ..................4: Comparison of (A) specific growth rate and (B) doubling time....1: Biomass concentration of C......................... ........ 38 Figure 4................................................. 24 Figure 3...................................... 33 Figure 4...........................................6: Examples of CSTR configurations ........................................................................... ... 2012) 42 Figure 4...........10: Effect of Mixing Speed on Cell Growth and Biomass Concentration ........ 45 Figure 4.... 47 Figure 4................7: Turbulence inside the stirred tank reactor at different mixing (Kazim....9: Steady state behaviour of C......................... vulgaris..................12: Biomass concentration vs dilution rate in a CSTR................... ..................................................... vulgaris in a tubular photo-bioreactor...8: Semi-continuous cultivation and nutrient consumption of C..................... 44 Figure 4............11: Effect of dilution rate on biomass concentration of Chlorella vulgaris in a CSTR............ 49 Figure 4.........................................Figure 4....................................... 50 x ...................... .......................................... ............................................. 66 Appendix D: Sample Calculations .................................. 67 xi .................................... 63 Appendix B: Calibration Curve for CO2 ...............................List of Appendices Appendix A: Equipment and Materials .............................................. 65 Appendix C: Determination of Dry Weight and Biomass Concentration ........................................................................ g/L concentration Rate of cell formation cells/day Time days ° Initial time days td Doubling time days V Volume liters Specific growth rate d-1 Greek Letters µ xii . Cell number. cell or biomass cells.Nomenclature D Dilution rate days-1 f Volumetric flow rate L/min mV Millivolt . g/L ° . ° Initial cell number. cell or biomass concentration cells. carbon neutral.. in comparison to plants and many microalgae strains are exceedingly rich in oil which makes them great candidates for the production of biodiesel. However. Using microalgae to produce biodiesel will not compromise production of human food. semi-continuous and continuous operation was investigated. Unfortunately. waste cooking oil and animal fat cannot realistically satisfy even a small fraction of the existing demand for transport fuels (Chisti. 2012).1 Chapter 1 1 Introduction The depletion of fossils fuel supplies and the contribution of these to the accumulation of CO2 in the atmosphere make it a priority to find renewable. pharmaceutical chemicals and pigments (used in food and cosmetics) (Becker. they have minimal nutrient requirements and are being used as source materials for a variety of products such as protein rich nutritional supplements. Biodiesel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels. . the high cost of production in bioreactors still remains a limitation and much research is required. transport fuels for environmental and economic sustainability. Biodiesel is considered as a possible substitute or extender of conventional diesel fuel. specifically the strain Chlorella vulgaris has shown a great promise as a source of oil. Microalgae are unicellular. a tubular photobioreactor and a CSTR. biodiesel from oil crops. There are many strains that have shown promise. 2007. They also grow extremely fast. 1994). photosynthetic microorganisms. GonzálezFernández et al. it is quite similar and compatible with conventional diesel fuel in its main characteristics. animal feed and other products as when oil crops are used. In this study. in batch. the production of microalgae (Chlorella vulgaris) in 4L flasks. In chapter 3. • Investigate the effect of different dilution rates on the biomass concentration of Chlorella vulgaris when grown in a Continuously Stirred Tank Reactor (CSTR). the pros and cons. their place and importance in the modern world. materials and methods listed including descriptions of the different photo-bioreactor configurations and parameters monitored. Chapter 2 contains a more detailed literature review relevant to the research such as basic information of microalgae. Chapter 1 contains a brief introduction to the research topic.2 1. 1.1 • Research Objectives Investigate the effect of nutrient media formulations on the growth of Chlorella vulgaris • Investigate batch and continuous cultivation of Chlorella vulgaris in different photo-bioreactor configurations. and how the research of this thesis fits into current research. analyzed and discussed. • Investigate the effect of different dilution rates on the biomass concentration of Chlorella vulgaris when grown in a tubular photo-bioreactor.2 Thesis Overview This thesis consists of five chapters. results obtained are shown. The final chapter (chapter 5) includes the conclusions that can be drawn from the results obtained as well as recommendations for future work. photo-bioreactor technology that is currently used for their proper cultivation. . • Investigate the effect of different mixing speeds on the biomass concentration of Chlorella vulgaris when grown in a Continuously Stirred Tank Reactor (CSTR). In chapter 4. Microalgae are generally more efficient than land based plants in utilizing sun light.3 Chapter 2 2 Literature Revi Review 2. 2011. Several studies have shown the potential of microalgae as a therapeutica therapeutical agents.2 Current Uses of M Microalgae The are multiple areas in where microalgae are used. enzymes) can be extracted. Figure 2. Figure 2. Scenedesmus. preparations containing Spirulina ulina have shown to help wound cicatrization. etc.1 shows an image of Chlorella vulgaris UTEX 2714.1:: Microscopic image of Chlorella vulgaris UTEX 2714 2. They are a type of eukaryotic cell and they contain similar organelles such as chloroplasts. the specifics will depend on the needs.1 Introduction on M Microalgae 2. CO2. which was used on the study mentioned in this thesis..containing preparations have been tested and .1 What are Microalgae Microalgae are microscopic photosynthetic microorganisms.1. Richmond. They can also grow in a variety of aquatic environments and can be grown without the he use of fertilizers and pesticides which results in less waste and pollution (Gouveia and SpringerLink.1. 2010) 2010). nucleus. It has been found that this strain of microalga can vary in diameter from 4-5µm (Chandhok. The whole algal biomass can be used as a source of protein or valuable chemicals (pigments. water and other nutrients which translate to higher biomass yields and higher growth rates. 2004) 2004). 1.. 2011. 1989).2: Various commercial applications of microalgae (Varfolomeev and Wasserman. 1994.4 shown effects on the treatment of certain skin diseases such as eczema and some algal compounds have shown inhibitory effects on the HIV virus (Becker. 1967..2.. Becker. 2011). This requires the development of alternative biofuels. 2006. non toxic. Gellenbeck. in the treatment of wastewater and as a potential source of oil for biofuels (Aslan and Kapdan. 2.1 Source of Oil for Biodiesel Reduction of CO2 emissions is expedient but even a 30% reduction (as agreed by some international legislations).2 shows a summary of various commercial applications of microalgae. it can be used in regular diesel engines. Clement et al. Biodiesel is a popular alternative to petroleum based diesel. as an additive for cosmetics. biodegradable and when burned. Microalgae are already used as a source of nutritional supplements. is still not enough to stabilize the CO2 levels into a “safe zone”. 1994. 2012) Figure 2. It can be prepared from . Feng et al. It is ecofriendly. of which biodiesel and bioethanol are the ones with the greatest market potential at the moment. Figure 2. Gustafson et al. it produces fewer emissions than its petroleum counterpart. due to its low sulphur content. (Bajpai and Tyagi. 2001) In Table 2. 2006.5 renewable sources like edible and non edible vegetable oils.. current biodiesel supplies from oil-production crops. Second generation biofuels (such as lignocellulosic and microalgal biofuel systems) have the potential to overcome many of the limitations. animal fats and even waste cooking oils. Biodiesel made from different vegetable oils has comparable properties as conventional diesels and can be used instead or as blended fuels in diesels engines.. 2008. 2000). Schenk et al. The most common concern with first generation biofuels is that as production capacities increase. As an example. . 2007. 12 million tons in 2007) current global oil consumption and cannot even come close to satisfying the existing and future demand for transport fuels. Srivastava and Prasad. (Lang et al. Srivastava and Prasad. than traditional oil crops. 2008.3% (approx. for biodiesel production. Schenk et al.1 it can be seen that some strains of microalgae are reported to produce 15-300 times more oil per area. traditional oil crops are only harvested once or twice a year. The cost of biodiesel could be reduced if one considers non-edible oils such as microalgae oil (Chisti. supplemented with small amounts of animal fat and waste cooking oil. Currently approximately 8% of plant-based oil production is used as biodiesel and this has already contributed to an increase in price of oil crops over the last few years. only account for an estimated 0. Microalgae also have a shorter average harvesting cycle of around 10 days. 2000).. so does their competition with agriculture for arable lands used for food production. 2008) . 2006.3 Algae (10g m day at 30% TAG) 12. in cold temperatures.500 49 0. Feng et al.3 2. . Biodiesel (L/ha/year) Land needed for cultivation (ha x 106) Global land needed for cultivation (%) Global arable land needed for cultivation (%) Soybean 446 10.5 206.. when growing in nature. 2012. 2006). Muñoz and Guieysse. In some cases pre-treatment maybe necessary since dark color and high fat content of the wastewater has been found to inhibit the growth of microalgae but this makes it possible to effectively use microalgae in the treatment of different animal farm effluents (Heidari et al. Chlorella protothecoides has been found to produce 29.000 406 2..6 Sunflower 952 5.932 73.190 4. 2005.892 2.7 20. 2008).4% of lipids (dry basis). 2006)..6 Table 2.. jathropa is mainly grown on marginal land.121 34. Miao and Wu.950 819 5.2. 2009.097 27. than biodiesel from vegetable oils and animal fats (Demirbas. 2010) 2. 2011.2% in a study where it was grown autotrophically (Demirbas.4 551. Muñoz et al.5 Crop Palm -2 -1 a If algal ponds and bioreactors are situated on non-arable land. 2011.5 (0a) Algae (50 g m-2 day-1 at 50% TAG) 98..4 Rapeseed/Canola 1.4 254..3 130 (0a) 5. Properties of microalgal oil indicate that its biodiesel has comparable characteristics and might perform better. Schenk et al..577 17.7 Jathropa 1. Subramaniam et al.1: Comparison of crop-dependent and algae biodiesel (Schenk et al.5 41. Miao and Wu. 2012. they can then help in the removal of pollutants including some carcinogenic chemicals. and up to 55. Different studies have shown Chlorella to have promise as a source for oil in the production of biodiesel (Ahn et al. Sirisansaneeyakul et al. TAG: Triacylglycerides Oil content in microalgae can vary widely from strain to strain..2 Wastewater Treatment Microalgae have been successfully used as part of the treatment of different types of wastewater.1. 2012. 2009.. Hodaifa et al. when growing different strains of Chlorella.5 and 9 (Richmond et al. 2010). Another study by (Zheng et al. which leads to confirm that microalgae utilized ammonium first and then the nitrate present in the inoculum. yielded 0. it was found that the maximum biomass concentration (4. Microalgae are known to grow more abundantly in nutrientrich (eutrophic) waters leading frequently to algal blooms (Schenk et al.. in the form of bicarbonate using an alkali NaOH.2 g/L of NaHCO3 (Yeh et al. showed similar results where the growth of Chlorella vulgaris. has shown to increase the biomass . 2011).5M or 4.6 g/L of biomass) at a concentration of 1. favored the growth of Chlorella vulgaris in a mixed culture with Spirulina platensis.2. A change of the concentration of nitrates (as KNO3) in the culture medium. 2011) A study published in 1982 showed that relatively low concentrations of NaHCO3 (0.. 2011) observed a depletion of ammonium (as NH4Cl) in artificial wastewater within 3 days of culture..24 g/L) was already high enough to promote growth (Sirisansaneeyakul et al..6 g/L. with NaHCO3 at a concentration of 40mmol/L (3. had little effect on the biomass concentration and lipid production possibly due to the fact that the lowest concentration tested (1.4 g/L) was similar on both cases when using 5% CO2 enriched air but the pH decline was more drastic in the case of the original TAP formulation (with NH4Cl) (Kumar and Das.36 g/L). In a comparison study of the growth of Chlorella sorokiniana. in TAP (Tris-AcetatePhosphate) media. The fixation of dissolved CO2.2 g/L) and bubbling with CO2 enriched air (1.67 g/L of dried biomass.. reaching its maximum (0.7 2. which ranged between 8. even after the depletion of ammonium. 2012). this being independent of the pH.1 Nutrient Composition of Media Media composition has an immense effect on both the growth rate and the final concentration of microalgae. The growth of Chlorella vulgaris was not limited. (Feng et al. showed an increase of biomass production by Chlorella vulgaris in a concentration range of 0.1-1. 1982). 2008).5%). Addition of NaHCO3 to Basal media.2 Factors Affecting Microalgal Growth 2.. with either 15g/L of NH4Cl or 1.5 g/L of NaNO3. as the concentration of NaHCO3 increased so did the biomass production. 3 shows the different forms of CO2 that can be available in water. With a photoperiod of 12h nearly doubling the microalgae biomass concentration in comparison with 3h. Photoperiods have a significant effect on the photosynthetic activity and growth rates in a typical microalgae cultivation system but it should be noted that excessive light exposure can lead to waste of electricity and cell growth inhibition....3 pH Initial culture pH can have a mayor on biomass concentration and lipid production of some strains of microalgae. 2.2 Light Duration of light periods is an important criteria to be considered in the design of a photobioreactor set-up.. Maximum growth rate and lipid production in the case of Chlorella was seen at a NaHCO3 concentration of 75ppm (75 mg/L) and with a concentration of CO2 of 4758ppm (Devgoswami et al. 2010). It has been found that growth rates and maximum cell density of microalgae increase proportionally to light period duration.. in comparison with the 14:10h (light:dark) (Jacob-Lopes et al. even at different light intensities (Amini Khoeyi et al. and 9h light periods (Lam and Lee. 1994. Jacob-Lopes et al.2. 2011). At the same time.. Figure 2.0. 2011).2. in comparison to the addition of CO2 alone (Aishvarya et al. the amount of each form that can be found in a solution at any given time will depend greatly on the pH . 1997) found that Chlorella vulgaris can tolerate a pH as low as 3. 2012). (Mayo. 2012. it can also cause the precipitation of some calcium salts if it exceeds a value of 9. 2012). Sirisansaneeyakul et al. 2009) 2.. 6h. An exception to this was discovered with a 12:12h (light:dark) photoperiod. in which higher productivities and maximum cell density values where obtained.8 production of Chlorella sp. high final pH (pH=11) caused negative effects on the growth of another strain of Chlorella vulgaris (Yeh et al. It has been noted that the lipid content of some microalgae increases with the supplementation of NaHCO3 to the media at different concentrations and bubbling with CO2 enriched air. 2009).0 (Becker. like the materials used for the set-up. source of light. either through a direct method. one must have a series of measurements. and photo-bioreactors. Closed systems provide a better opportunity meet specific demands and in the control and optimization cell growth parameters. 2007). how will the algae circulate through the reactor. 1994). 1994) 2. It is important to maintain pH within an adequate range to avoid the loss of carbon dioxide present in the media (Becker. with light supplied by the sun. and how to control other parameters such as pH. temperature and nutrient concentration in the media (Andersen. 2. 2012) found that the most adequate growth was under indoor conditions and utilizing inorganic nutrient media. Cell number should be counted.3: Carbon dioxide equilibrium in water (Becker. In the case of photo-bioreactors. many design considerations need to be made depending on what the end goal is.9 of the solution. Chisti. the assimilation of CO2 by the growing microalgae causes a shift on the equilibrium shown on Figure 2. Mainly outdoor ponds.3 which results in an increase of pH values due to the excretion of OH.by the algae into the media. Figure 2. There are basic design features that should be considered regardless of the configuration. which can be outdoor or indoor and light can be supplied by electric lights. as through light microscopy with a hemacytometer. There are various configurations that have been built and tested for the growth of microalgae. how to provide CO2.3 Microalgae Cultivation Different approaches can be taken when looking to grow microalgae in large volumes.3. or indirectly through biomass concentration (as dry weight) or optical . A study on Chlorella vulgaris by (Lam and Lee. that will permit the calculation of the rate of change in biomass concentration. In a poorly buffered system. Species control is better achieved under closed conditions that are very common in laboratory setting.1 Microalgae Growth Kinetics To estimate growth rates. at different times. 2005. Logarithmic death phase C BIOMASS CONCENTRATION D B A TIME Figure 2. 1994) Under a typical homogenous batch regime (in a closed system). the number of living cells. doubles regularly with time (Bailey and Ollis.4: A. are not always clearly defined. equations (2. in this phase. 1994).4. or (2. During lag phase (phase A) the microalgae cells are adapting to the new environment conditions. Becker. shown in Figure 2. illustrated in Figure 2. Becker. Exponential growth phase (log phase) C. as long as this measurements correlate linearly with the number of cells (Andersen.2) describe cell growth. at the end of the lag phase. their length or slope might change according to the culture conditions. 1977. this is the exponential phase (phase B). microalgae will pass through the following growth phases. 2005. Adaptation (lag phase) B. the cells are well adjusted and then start to multiply rapidly.1) and (2. Stationary phase D.10 density.1) .4: Microalgae batch growth profile The individual phases. During this period. ° is the cell or biomass concentration in the feed stream. 1 is the culture volume and is the rate of cell formation.2) Where and ° are the number of cells (or cell concentration) at different times and ° .5) !" #$ +# .11 ° or ° (2. is the cell concentration inside the system.6) to obtain the following (Bailey and Ollis. where ° is the initial time and is the specific growth rate. In an open system.4) or can be used to characterize the state of the cell population.6) Where $ is the volumetric flow rate.-%# . media with fresh nutrients is added in at the same rate that medium is withdrawn from the culture.1) the following is obtained: ° or ° ° (2. 1977): . If it is assumed that the growth rate is much higher than cell death rate and cell death is neglected. this keeps growth parameters and cell concentration at a constant level. then it can be substituted in equation (2. From the integrated form of equation (2. The following mass balance can be applied to any component of the system: !" #$ ! %%# # &'&"( – !" #$ "(#*! $#( &'&"( (2.3) it can deduced that the time required for cell population to double ( ) is given by: During the exponential phase ln 2 (2. if µ denotes the specific growth rate ( ⁄).3) ° From equation (2.%/% &'&"( 0 The above can be reformulated as: $° $ 1 0 (2. 3. Table 2.2 (2. equation (2.7) The ratio of the volumetric flow rate. .9) (2.12 3° 3 (2. so that: 3 $/1 (2. meaning the algal cell concentration is constant in the system.9) and (2. inclined at different angles) (Wang et al.10) are fulfilled (Becker. Tubular PBRs are the most popular configurations and can be arranged in several different patterns (straight. vertical.10) Photo-bioreactor Design There are many possible photbioreactor (PBR) configurations. spirals) and in different orientations (horizontal.2 shows a summary of research performed in various PBR configurations using different microorganisms and obtaining a variety of results.. 2012). bent.8) When the liquid feed stream to a continuous culture consists only of nutrients. at which fresh medium is added to the culture. 1994). ⁄ 0 μ 3 2. so that ° 0. is referred to as the dilution rate 3. 03-0.0 ± 0. (L) freshwater 25±2 2 (for pH control) 0.127 13.8 1 0. Chlorella vulgaris obliquus D..23 -1 Lipid productivity/ Diluted Manure 0.7g L d Biomass Productivity Biomass Concentration (g/L) 1. 2011) (Wang et al. (Hulatt and Thomas.4 d-1 0. tertiolecta Type Artificial Wastewater Media Media Supplement Glucose .2 1.5 1.5 % dry weight content Dilution Rate pH 0.13 Table 2.) tubular PBR Design BG11 0. 2011) Scenedesmus (Henrard et al.2: Summary of continuous and semi-continuous cultivation of microalgae Author (Feng et al.28-.8 (initial) 7. NaNO3 2 1 -1 1.5-1..89 (cont.076-0.6 g/L NaHCO3 absorbed by NaOH solution Working Vol.5 7. NH4Cl cyanobacterium freshwater freshwater Fitzgerald media Modified Bold 3N Jaworski 15% CO2 3 x Reg.3 d-1 . 2012a) Species Chlorella vulgaris Thermosynechococ cus sp Chlorella minutissima.05-0. 2012) (Tang et al.4 -1.64 d-1 Not specified 8-10 10-11.726 2..72 (batch) Tubular Light Source 30 Temp.5-3. 2010a) Cyanobium sp.2 Not constant 0... 2011) (Su et al.5 1.38 CSTR Fence + Tank Tubular 4L flasks Circular LED Sunlight -1 25 4 % CO2 Growth Rate (d ) 37% Fluorescent lamp 30 to control pH 0. (ºC) 3–6 500 1. usually there is a sparger to allow for smaller an multiple bubble formation for better mixing of the culture.... therefore. it is possible to reduce their diameter to allow for a higher surface-volume ratio. Mechanical agitation in photo-bioreactors is known to enhance radial mixing but it can cause damage to the algal cells. 2006. 2009). optimal O2 removal and it is gentle enough to avoid shear stress damage to the cells even a high superficial gas velocities (Sobczuk et al. Both configurations have been extensively studied for the production of microalgae (García Camacho et al. 1999.. 2005.3. 2002. Air agitated bubble columns and airlift bioreactors have been studied for the culture of large amounts of microalgae but there are restrictions in height since it could negatively affect gas transfer and increase residence time of O2 generated during photosynthesis. tubular photo-bioreactors are simple and very easy to clean (Wang et al. 2007.. 2012. Wang et al.. Vega-Estrada et al. 2012) A typical vertical column or tubular PBR (Figure 2. 2012).5 B). Sánchez Mirón et al.14 2.3 Tubular Photo-bioreactors Tubular photo-bioreactors with shallow light paths are commonly used to grow photosynthetic microorganisms. Besides excellent mixing. Xu et al. air bubbling from the bottom of the column (which is common in tubular PBR) is preferred since it enables efficient CO2 utilization.. Kumar and Das. which can lead to cell growth inhibition.. in some cases there is an added internal column which creates a airlift photo-bioreactor (Figure 2. Krichnavaruk et al. .5 A) consists of a transparent column with air inlet at the bottom. 4 Continuously Stirred Tank Reactors Stirred-tank reactors.. example (B) shows a more complex reactor. they are easy to model and systems are available that could easily monitor and control the main experimental parameters. 2005). being operationally and structurally simple.15 Figure 2. . one of possible importance in microalgae cultivation is the mixing speed. example (A) shows a simple flask on top of a stirring plate where the stirring is done by a magnetic bar. On Figure 2. There are many parameters that could be considered when doing an experiment on a CSTR.5: Tubular photo-bioreactor diagram 2. are ideal devices for cultivating different types of cells.3. CSTR can be beneficial in preliminary studies for ideal growth conditions and determining different parameters. a continuously stirred tank reactor can come in different capacities and different configurations (Muñoz et al.6. two examples can be seen. including microalgae (Yang and Wang. 1992). Whether it is a batch or continuous run. where the stirring is done by impellers and bubbling air. the ideal mixing speed will vary according to species and the chemical that is being investigated.200 rpm. 2012.16 Figure 2. In a study of Chlorella sp... 1999. 2012. 2012.. 2006).. it was found that chlorophyll was more efficiently produced at a mixing speed of 250 rpm (Funahashi et al. ... 2011. have shown similar results when used to grow microalgae for different purposes (Muñoz et al. Mazzuca Sobczuk and Chisti. 2010. 1999) . But again. Hodaifa et al..4. 1999. The maximum mixing speed that microalgae will tolerate without cell damage varies and is very dependent on the species (Córdoba-Castro et al.3. Sobczuk et al. if too high it could not only be impairing cell growth but it could be damaging in other ways as by causing the leakage of important chemicals from within the cell (Funahashi et al. 2005).1 Effect of mixing speed It is important to know the effect of the agitation speed on microalgae. Both CSTR and tubular photo-bioreactors. 2012. Mitra et al.. 2006) CSTR are used to grow microalgae at different mixing speeds within the wide range of 100 – 1. Sirisansaneeyakul et al..6: Examples of CSTR configurations 2. Funahashi et al.. Ohira et al. Sobczuk et al.. Filali et al. Tang et al. although this is almost ten times lower a growth rate than the maximum specific growth rate of 0.33 d-1.1 Effect of Dilution Rate Generally.135 g/L (Mazzuca Sobczuk and Chisti.17 2. with the productivity in the latter being 130 mg L-1 d-1.02 g/L at the dilution rate of 0. 2012b. 2010). this is a correct assumption when the are cultures that can grow to high densities (Andersen. of the microalgae Scenedesmus sp.08 to 0.. This is the general case for various microalgae species grown in continuous or semi-continuous systems. 1977) As mentioned in a previous section.5 Batch and Continuous Cultivation A batch reactor is a closed system with respect to exchange of matter with its environment. Wang et al. while a continuous reactor is an open system with mass exchange across its boundaries (Bailey and Ollis.. found that it could reach a maximum biomass concentration of 2. . 2010b) 2. In contrast. 2012) found that the biomass productivity.3.046 h-1 reached during the batch study. they tend to show greater productivity when grown continuously or semicontinuously versus when grown in batch (Cerón-García et al. 2012.3.. was two times greater when growing in a continuous chemostat than in batch cultivation. increasing the dilution rate of a system will lead to an increase of biomass concentration and cell productivity.005 h-1 (at 25 ºC). the steady-state concentration of cells in a continuous culture can be determined by the dilution rate.. with the maximum productivity reached at the dilution rate of 0. 2012b) found that the productivity of Chlorella minutissima varied from 39 to 137 mg/L/day (dry mass) as the dilution rate increased from 0... Sun et al. the maximum biomass concentration reached in the latter case was only 0. (Tang et al. 2011. 2009.5. 2005) (McGinn et al... a study where the microalga Choricystis minor was grown continuously.64 d-1. they show great potential as a source for different types of biofuels without competing with food sources and are easy to grow in photo-bioreactors. Photo-bioreactors provide an easy way to grow microalgae in large volume and they come in different shapes. Tubular and continuously stirred tank reactors are choice photo-bioreactors designs for studying and optimizing the different parameters that affect microalgae growth. Continuous cultures in photo-bioreactors have shown to improve overall biomass production of microalgae hence the importance of studying.18 2. used in cosmetics and as a source for oil for the production of biodiesel. including Chlorella vulgaris. sizes and configurations. Various microalgae strains within the genus Chlorella. not only their growth in different photo-bioreactor configurations but also the effect that dilution rates can have in a microalgae system. With few additions. any photo-bioreactor can be converted to run continuously.4 Importance of This Study Microalgae are already widely used in many commercial areas. as a source for nutritional supplements. . each of which have their own advantages and disadvantages. have shown promise in treating waste water. 2. the formulation shown in Table 3.2 Growth Media 3.1 Microalgae Strain The microalga Chlorella vulgaris UTEX 2714 was used for this study. 2005).19 Chapter 3 3 Materials and Methods 3. as reported by (Andersen. It contains macro and micro nutrients necessary for the growth of microalgae.1 Bold’s Basal Media A modified version of Bold’s Basal Medium. The strain was obtained through the Culture Collection of Algae from the University of Texas at Austin. The stocks solutions were prepared as needed and stored in the fridge until final media preparation. was used in most of the experiments. 3. .1. 42 1 8.1: Modified Bold’s Basal Media (Andersen.00 1 4.1 Stock Solution (g/L) Quantity Used (ml) 25.50 2. 1907). it was sterilized with a PALL.50 10 10 10 10 10 10 1 50.6g/100ml H2O (Seidell.2. .50 7. 9.82 1. 0.44 1. NaHCO3 would also form precipitates if autoclaved with the other media ingredients.98 1 1 11.1.57 Addition of NaHCO3 NaHCO3 was added to the regular Bold’s media formulation.00 31. 2005).45µ Acrodisc syringe filter. therefore it was added after the media was autoclaved. Component Macronutrients NaNO3 CaCl2·2H2O MgSO4·7H2O K2HPO4 KH2PO4 NaCl Alkaline EDTA Solution EDTA KOH Acidified Iron Solution FeSO4·7H2O H2SO4 Boron Solution H3BO3 Trace Metal Solution ZnSO4·7H2O MnCl2·4H2O CuSO4·5H2O 3.50 7.00 2.50 17. The solubility of NaHCO3 in water is very low.20 Table 3. 42mg 2.98mg 1ml ---- ---- 11.2.2.8mg ---- 1g ------250mg 25mg 75mg 75mg 175mg 25mg ------- ---200mg ---250mg 25mg 75mg 75mg 175mg 25mg ------- ------- ------- 63.021mg ------8. Components* Macronutrients Proteose Peptone NH4Cl KNO3 NaNO3 CaCl2·2H2O MgSO4·7H2O K2HPO4 KH2PO4 NaCl FeSO4·7H2O FeEDTA Alkaline EDTA Solution Na2EDTA KOH Acidified Iron Solution FeSO4·7H2O H2SO4 Boron Solution H3BO3 Trace Metal Solution H3BO3 Na2MoO4 ZnSO4·7H2O MnCl2·4H2O CuSO4·5H2O C30 Proteose PCS-2 ------5g ------2.68mg 0.25g ---2.44mg 1.021mg 2.5g 100mg 1.11mg 1.21 3.11mg 1. Table 3.9mg 31mg ------- ------- 4.2: Other Media Formulations (Perez-Garcia et al.09mg 0.68mg 0.2 Other Media Formulation The different media formulations used in experiments are listed in Table 3.81mg 0.81mg 0. 2011).82mg 1.09mg 0..57mg . 3 Inoculum and Strain Maintenance Previous to every photo-bioreactor run. and more than one experiment could be run at the same time. . air exchange was allowed through foam stoppers and flasks were placed away from direct sunlight. Culture samples were taken through the sampling port with a sterile syringe. depending on the experiment. which allowed gas exchange in small volume flasks (50-250ml). Light was provided by placing the flasks by a sunny window.1 4 L Conical Flasks Figure 3. Air was supplied through a fish tank air pump (Top Fin 4000. Strain maintenance was done only in small culture volumes (25-100ml). Bold’s or a combination of Bold’s and Proteose media was used. The inoculum set-up consisted of 4L flasks with a 3L working volume. Canada).1 shows the set-up for 4L flasks with a working volume of no more than 3. Air was supplied by either foam stoppers. PetSmart.22 3. PetSmart.4 Photo-bioreactor Set-up Three different photo-bioreactors configurations were used for the experiments with Chlorella vulgaris. a separate inoculum was prepared.4. a tubular and a continuously stirred tank reactor (CSTR) 3. The growth media was either Bold’s or a combination of Bold’s and Proteose media. or through a fish tank air pump (Top Fin 4000. These configurations were 4 liter conical flasks. This configuration was easy and quick to setup. Canada) and stone sparger for larger volume flasks (1-4L). The flasks were positioned by a window to provide sunlight. 3.5 L. to slow down cell growth. 23 (1) Stone sparger. (3) Sampling port. depending on the working volume.2 Tubular Photo-bioreactor The general diagram for the tubular photo-bioreactor is shown on Figure 3.0x10-2 m.4.1 m and a wall thickness of 1.2. (2) Air filter. Pictures of the different volume set ups can be found on Appendix A. The height of the reactor varied from. . 3.1: 4L Flask Set-Up. The reactor consisted of tubular sections of different lengths made of plexiglass. (4) Air exhaust Figure 3. all set-ups had an inside diameter of 0. 06 m respectively. The O.2.D.09 m and 0.2.3: Diagram of Ring Sparger 3.2: Tubular Photo-bioreactor Diagram 3.3) was used on the tubular photo-bioreactor experiments. with aeration holes measuring 0.1 Sparger A ring sparger (Figure 3.15x10-2 m in diameter.4. were 0.2 Batch and Semi-Continuous Experiments For samples taken during the batch stage of the experiment (before steady state was reached) a sampling device consisting of a pipette attached to a syringe through a piece of .D and I.4. Figure 3.24 Figure 3. 4. (4) Ring sparger .25 tubing was used.4. all other analysis were done from samples removed from the photo-bioreactor. For all different dilution rates. a picture can be found in Appendix A. 3. as shown in Figure 3.asp?action=download&fileid=5700 Figure 3.4: CSTR Diagram. During continuous runs ports (1) and (3). directly from a valve at the bottom of the column. it allowed for easy sampling through the top of the bioreactor .ch/puebersicht.3 Continuously Stirred Tank (CSTR) Photo-bioreactor A Bioflo 3000 fermentor (Eppendorf New Brunswick. Mississauga. (2) 6-blade Rushton impeller. Source: http://www. were used for inlet of media and outlet of culture respectively.igz. Maximum capacity was of 14L with a maximum working volume of 10L. Temperature was measured directly from the culture in the photo-bioreactor with a digithermo digital thermometer (VWR International. (3) Culture harvest tube.4 shows a diagram of the CSTR. ON) was used to study the effects of mixing speed in microalgae growth. Figure 3. every two days. (1) Media inlet. a set volume of culture was taken out of the PBR. Canada). 2 and 3 (as shown in Figure 3. pH. Quebec.3.4. Canada) were utilized to maintain a steady state. Mississauga. mixed and regulated with a custom made multi tube rotameter (OMEGA Engineering. two low flow micro pumps (VWR International.5. They were calibrated and adjusted as needed to obtain different dilution rates. Canada) for all tubular and stirred tank photo-bioreactor experiments. . Canada). The lamps operated to provide a 12hr light supplementation to simulate the regular night/day light cycle.4. 3. The data collected was extracted every few days and transferred to an excel file.2 Batch and Continuous Experiments Samples were taken every 1-2 days during the batch period For the continuous runs on the CSTR.4. 3. mixing speed and temperature. regular fluorescent light bulbs were replaced with 21watt daylight light bulbs. The settings of the software were arranged so that readings were taken every minute for the first hour.4.1 Monitoring of Parameters Track & Trend Biocommand (Eppendorf New Brunswick Scientific.3. ON) was used to track dissolved oxygen. at the start of every experiment. 3. Quebec.4. In all cases.26 3. which simulated the glow of natural sun light.5 Lighting There was different lighting for the different photo-bioreactor settings. Light intensity readings were taken on points 1. Canada). and then every hour after that. 3. on the outside surface of the photobiareactor. light intensity was measured with a light meter (VWR International.1 Tubular PBR The tubular photo-bioreactor was situated by a large window therefore light was provided by the sun but supplemented with two tubular fluorescent lamps (Illume.4 Air Supply Pressurized air and CO2 were supplied.5). Four 12. Light intensity readings were taken by the outside surface of the photo-bioreactor. on both sides of the photo-bioreactor as shown in Figure 3.5.4. 8watts fluorescent lights (Bazz. Figure 3.2 CSTR No direct sunlight was provided in the case of the CSTR. The lamps operated to provide a 12hr light supplementation to simulate the regular night/day light cycle. Light Set-Up on Tubular Photo-bioreactor 3.27 Figure 3.5.5in. directly in front of where each pair of lamps were placed. Light Set-up On Stirred Tank Photo-bioreactor .6.6. Quebec. Canada) were placed. in pairs. ). If it pH was out of range.7 Determination of Dry Weight and Biomass Concentration To determine the dry weight of every sample would not have been efficient. . therefore.1 Cell Count Cell count and cell condition were determined through light microscopy (Leica.28 3. and weighted to obtain the dry weight of the sample. Germany) with an improved Neubauer phase hemacytometer (Hausser Scientific. 3. a diagram of the hemacytometer counting grid is presented in Appendix A. Except for the CSTR runs where it was measured in-line.).6. previously sterilized water was added to make up for any evaporation loss. 3. was created using the following procedure: 1. dried in an oven at 105 °C for 24-48hrs until the weight of the filter was unchanged. 2 samples of 50ml of Chlorella vulgaris culture were measured 2. first.).6 Analysis of Samples 3.5 Sampling of Photo-bioreactor For sampling in the case of all batch and semi-continuous runs. it was adjusted with either a 1N HCl. The first sample was filtered through a 42.S.5mm round GF/C glass microfiber (Whatman. U.. the PBR was left undisturbed for about 5 to 10 minutes. U. 3. absorbance at 686nm was taken. the calibration curve. then the sample was taken. 3. The second sample was used for two-fold serial dilution.K.S. 5N HCl or a 5N NaOH solution.6. U. show in Appendix C.2 pH Measured in every sample with a pH meter (Beckman Coulter Inc. 1. 5 ml of the standard carbon dioxide solution was added in the increments of 1 ml. The solution was stirred and 0. which at the same time was connected to a computer that recorded and stored all reading through the Accumet XLComm (Fisher Scientific. it entered the probe in the form of carbonic acid (H2CO3). Fisher Scientific. Once the mV value stabilized. The purpose of using 0. Ottawa. it was zeroed. Afterwards. This change in the H+ ion concentration was interpreted as potential difference and the reading was displayed on the pH meter in millivolts.29 4. The CO2 electrode was not part of the Bioflo 3000 bioreactor original set-up.1 Checking Slope of CO2 Probe The slope of the carbon dioxide probe assured the proper operation of the probe. 3. As soon as the probe was immersed inside the solution the negative mV values begin to decrease in magnitude. In order to check the slope of the electrode. The theoretical weight from the serial dilutions of the second sample were correlated to their absorbance reading. which brought a change in the H+ ion concentration in the refill solution of the electrode. Higher mV values meant higher concentrations of dissolved carbon dioxide in the sample. The CO2 electrode was connected to a pH meter (Accumet XL-50. When dissolved carbon dioxide passed through the membrane. 5 ml of a sodium citrate buffer solution were added to 45 ml of distilled water in a 100 ml beaker. Laval.5ml and 5ml of the same solution was to obtain the change in mV when the CO2 concentration in the sample increased ten-fold.8. ON). Quebec) was used to measure the concentration of dissolved carbon dioxide during experiments. . 3. Omega. it was incorporated through a specially designed holder that fit into one of the bioreactor’s ports.5 ml of standard carbon dioxide solution (1000 ppm as CO2) was added. A slope 30-62mV assured proper operation of the probe. Ottawa.8 Determination of CO2 Concentration An ion selective carbon dioxide electrode (Model no: ISE-8750. as soon as the mV value was stabilized it was determined as the slope of the probe. ON) software. The resulting equation was used for calculating the theoretical biomass concentration of experimental samples. 30 3. were used to analyze the concentration of phosphates in the filtered sample. The equation and graphs are shown on Appendix B. was created and the equation generated was used to convert the mV readings into ppm of CO2. A DR 2800 spectrophotometer (HACH Company. correlating the potential difference (mV) vs.) and appropriate test kits were used for the analysis of nutrients (nitrates and phosphates). Three standardized carbon dioxide solutions (10.9 Nutrient Analysis For nutrient analysis. Method 8114 of the DR2800 spectrophotometer procedures manual was followed.9. U. UK) and frozen at -20ºC for later analysis. 100 and 1000 ppm CO2) were used.8. 3. 3.45µ syringe filter (Acrodisc. Cornwall. Method 10206 and method 10020 of the DR2800 spectrophotometer procedures manual was followed.1 Nitrates TNTplus 836 vials (HACH Company. U. U. The reading of the 100ppm solution was used as a zero and a logarithmic graph. At the day of nutrient analysis. Maximum theoretical and expected phosphate concentration was calculated and the filtered sample was diluted as needed. high range (1-100 mg/L PO4-3). samples were thawed out and allowed to reach room temperature.1. an aliquot of no less than 7ml was taken and filtered through a 0. Colorado.9.S.) high range (0.S.9-133 mg/L NO3).2 Calibration Curve for CO2 Probe A calibration curve for the carbon dioxide probe was created before the start of every CSTR experiment. Colorado. .2 -30 mg/L NO3-N or 0. Maximum theoretical and expected nitrate concentration was calculated and the filtered sample was diluted as needed.S.S. Colorado. the methodology listed for each kit was followed.) for reactive phosphorus (orthophosphate). 3.2 Phosphates Test’N Tube vials (HACH Company. Colorado. Concentration (ppm as CO2). U. were used. PALL Corporation.) high range (5-35 mg/L NO3-N or 22-155 mg/L NO3) and Test’N Tube vials (HACH Company. 1.15 g/L). The reason for this experiment was to confirm a previously reported study on the effect of NaHCO3 on the growth of newly isolated strain of Chlorella vulgaris.31 Chapter 4 4 Results and Discussions 4. the growth of Chlorella vulgaris in regular Bold’s media and in Bold’s media with added sodium bicarbonate at a concentration of 1. 4. the control had a lower but steadier growth rate of 0. the control’s biomass concentration (0. At the end of the experiment.53 d).1 Modified Bold’s Media with NaHCO3 It can be observed in Figure 4. the culture with added NaHCO3 grows more rapidly at the beginning but then its growth decreased as the control’s (Bold’s media) continued to increase. High flocculation of the microalgae was observed in the culture with added NaHCO3.23 g/L) was higher than the one for Bold’s media with added NaHCO3 (0.345 d-1 (td = 2. 2010). The total working volume for each culture was 3.2 g/L was investigated.1 Batch Cultivation in 4L Flasks First.2 g/L of NaHCO3 in an artificial wastewater media (Yeh et al.1. It was hypothesized that the addition of NaHCO3 may serve as an additional supply of carbon for the microalgae hence eliminating the need to supply the air with gaseous CO2.. this study showed a beneficial effect with a concentration of 1.125 d-1 (td = 5. The culture with NaHCO3 presented an initial growth rate of 0. . growth decreases at around the seventh day and even biomass concentration decreases. that both cultures have a close biomass concentration during the first couple of days.01 d) but as mentioned before.5L with 20% of that being inoculum. 2 shows that the pH increased drastically in the culture with 1. Generally. vulgaris in 4L flasks. 2010).. Bold’s with added NaHCO3 (1.anions from the media and if it reaches values above 9. 2010) did find a positive effect of NaHCO3 on Chlorella vulgaris growth but they also found that higher concentrations of NaHCO3 caused a higher final pH (pH=11) which resulted in a detrimental effect on biomass concentration.25 0. pH is expected to increase due to depletion of NO3. The decrease in growth rate and the flocculation of cells observed could be due to the change in pH.10 Control NaHCO3 0. The study done by (Yeh et al.15 0. . It is possible that the strain used in our study has different sensitivity levels to NaHCO3 than the one used in the study by (Yeh et al.05 0.32 0..0. 2005). Figure 4. the pH of the control culture did not go above a value of 8. it can cause precipitation of various salts which can lead to nutrient deficiency for the microalgae (Becker. Bold’s media (Control) vs.1: Biomass concentration of C.20 0.00 0 2 4 6 8 10 12 14 16 Days Note: Error bars represent standard deviation from the average biomass. the pH values reached also contradict the expected effect of added bicarbonate ions acting as buffers (Andersen.30 Biomass Concentration (g/L) 0.0 throughout the whole experiment. The pH of the NaHCO3 culture was way above the range which most fresh water microalgae prefer (5-7).2 g/L) Figure 4. 1994). which reaches a value above 10.2g/L NaHCO3. 5L with an inoculum concentration of 8. because of the relatively high nitrate content (5g/L of KNO3) and the enriched air with 5% CO2 (v/v). In the case of C30 media.7%. It was expected that Chlorella vulgaris would grow well in both C30 and PCS-2 media. C30 media has been used to grow Chlorella sp (Perez-Garcia et al. For the experiment with C30 media. 2011).2.33 11 10 pH 9 8 Control NaHCO3 7 6 0 2 4 6 8 10 12 14 16 Days Figure 4. The results with PCS-2 media were also expected to be quite favorable due to the similar formulation as Bold’s media and with added source of nitrogen as ammonium chloride (200mg/L of NH4Cl) since microalgae seem to prefer nitrogen in the form of ammonia and have also been successfully used to remove ammonia from artificial an real . The reactors were placed by the window so sunlight was the main light source and the air was enriched with 5% CO2 (v/v).. 4. Li et al. 2008).2: Change of pH with the addition of NaHCO3 4. since it has been proven that nitrates is a preferred form of nitrogen by microalgae and appear to be significant for adequate growth (Lam and Lee. the total working volume was of 3.1 Batch Cultivation in C30 and PCS-2 Media The tubular PBR configuration previously mentioned and shown in Appendix A was used.. 2012.2 Batch Cultivation in Tubular PBR. 2010) has found that Chlorella sp. 2011). 2010.0 0. Chandhok.. 2011. 2012. and Scenedesmus sp.13 0. The stunted cell growth in C30 media could be due to different reasons...3. with an overall negative growth rate.2 PCS-2 Bold's 0. The relatively low inoculum concentration. it has been found that CO2 inhibition levels can vary depending on the microalgae specie (Lee and Hing. a comparison of the change in .7 Biomass Concentration (g/L) Biom ass Concentration (g/L) 0. Park and Craggs. A). There was an initial increase of the cell number but after just a couple days. Rados et al. in the form of FeCl3/EDTA. the source of iron (FeSO4·7H2O) could be not as biologically available for the algae..11 0.34 wastewater (Aslan and Kapdan.4 0. could have also been a factor affecting the growth (Mattos et al.3: Chlorella vulgaris growth in C30 (A) and PCS-2 (B) media.5 0. the stage of growth the inoculum was in. inhibition by CO2 is quite possible. of Chlorella vulgaris cultures (Liu et al. in comparison with the control (no iron added). 1975) and (Westerhoff et al. so in the case of C30 media. González-Fernández et al. 2006.1 0. from a Sonoran desert mineral spring (Mexico). 2008). or the concentration could be too low. 0. can tolerate a CO2 concentration of up to 20% (in gas). 1989. the count steadily decreased (Figure 4.. It has also been found that the addition of iron.6 0. Xiao et al.3 0. due to the lack of a chelator as EDTA.12 0.. No real improvement was noticed with PCS-2 media when compared even with the slowest growing microalgae experiment in Bold’s media (B6) which contained only nitrate as a source of nitrogen. The experiment was stopped after 7 days.. It appears that C30 was not an appropriate media for this strain of Chlorella vulgaris.10 0. increased the final cell density and lipid production.09 0 2 4 Days (A) 6 0 2 4 6 8 10 12 14 Days (B) Figure 4. 2012). 717 g/L for experiment B6. Experiments with Bold’s media in a tubular PBR were labeled from B1 to B6.0-7.0 (Moheimani. in the tubular photo-bioreactor set-up.. This results are in line with what is normally seen an expected in experiments where photosynthetic organisms are involved. Experiments B4. In both the C30 and PCS-2 experiments a lower average pH value can be seen.. Experiments with no added CO2 reached their maximum biomass concentration after 14-16 days.54 and 5. B2 and B3 all had very close specific growth rates (µ) values.3. 2012. it is commonly seen that in microalgae cultures. at 5.6 and Table 4. than the Bold’s media experiment values (pH=6..2 Batch Cultivation in Bold’s Media Multiple experiments were conducted. with various experimental specifications.32 d-1. Despite all the differences in experimental conditions.5). In Figure 4. 2012.2 d-1) which were not supplemented with CO2 enriched air. experiment B1 was performed in April (early spring). Experiment B1. B1but exp. The specifications and a compilation of results are shown in Figure 4.29 d-1 to 0. 4..33 respectively. and other microalgae have been found to be higher around pH values of 7. with no additional CO2. B2 and B3 all were aerated. at the same flow rate. with CO2 enriched air. The lower specific growth rate within the . Experiment B2 and B3 had a higher inoculum percentage than exp. B5 and B6 (from 0. a similar flow rates.033% (Andersen.2. (Mayo. 2010. since the normal concentration of CO2 in air is very low at around 0.547 g/L for experiment B1. 1997) found that Chlorella vulgaris can tolerate a pH as low as 3. B. that have an added source of carbon (organic or inorganic) there will be a positive effect on cell growth (González-López et al. B5 and B6 were supplied with ambient air. 2011). 2010). experiment B2 and B3 were performed in June and July (late spring/summer) respectively. Figure 4. Yeh et al. Yu et al.1.0 but biomass and lipid productivities in Chlorella sp. experiments B1. 2005). B1 had a higher initial biomass concentration.5 it can be observed that experiments aerated with CO2 enriched air reached their maximum biomass concentration after 6-9 days of the start of the experiments with the highest biomass concentration at 0. the highest biomass concentration was 0. from 0.5. Westerhoff et al.35 biomass concentration with time can be seen in Figure 4.15 d-1 to 0. which are higher than those µ values for experiments B4. Jun Jul Sep Jul Jan May Working Volume (L) 3. this could possibly be due to inhibition by longer light periods usual in the time of year when the experiment was performed.36 tubular photo-bioreactor experiments with Bold’s media. 2001) Table 4.00 6.547 0.54 7.680 0..717 Air Flow Rate (L/min) -1 ..32 0.5 Specific Growth Rate.5 1. Temperature (ºC) 21.3 6. Lee and Lee.298 0.0 16.7 6. was that of experiment B6. light intensity (lux) 2680 2350 1860 --- 4160 3800 Month of exp.17 3.98 4.3 23 24.202 0.444 0.30 Avg.8 10 10 Inoculum Concentration (%) 20 38 30 21 20 20 Additional Light Source No Yes Yes No Yes Yes % CO2 2. µ (d ) 0.0 22.54 7. 2012. Biomass Conc.445 0.42 2. it has been found that different light/dark periods do have an effect on microalgae growth.5 2 2 No No No 2 2 2 2 1.233 0.154 Doubling time (days) 2.32 2.8 21.8 21. Jacob-Lopes et al. nutrient removal and oil productivity (Amini Khoeyi et al.50 Max. (g/l) 0.1: Comparison of Chlorella vulgaris growth in Tubular PBR with Modified Bold’s Media Experiment Number B1 B2 B3 B4 B5 B6 Avg.1 Avg.26 2.10 6.550 0. 2009.307 0. pH 6.40 7. 30 0.20 0.4: Comparison of (A) specific growth rate and (B) doubling time. Figure 4.1 for more details.35 5 CO2 CO2 0.00 0 B1 B2 B3 B4 Experiment (A) B5 B6 B1 B2 B3 B4 Experiment (B) *See Table 4.10 No CO2 4 Doubling Time (days) Specific Growth Rate (µ) No CO2 3 2 1 0. For experiments performed in a tubular photo-bioreactor with Bold’s media.25 0.05 0. B5 B6 .15 0.37 0. 6 0. B3 0.5 0. B1 Exp. B6 0.0 0.3 0. B5 Exp. .1 No CO2 0.8 CO2 0.1 for more details. B2 Exp.4 Exp. Figure 4.2 0.0 0 2 4 6 8 10 0 2 4 6 8 Days Days (A) (B) 10 12 14 16 *See Table 4.38 0.2 Exp. For experiments performed in a tubular photo-bioreactor with Bold’s media.4 Biomass Concentration (g/L) Biomass Concentration (g/L) 0. B4 Exp.5: Comparison of change in biomass concentration of experiments with (A) CO2 enriched and (B) no added CO2 in air. 2 shows the results and parameters for the exponential growth phase of both previously mentioned experiments.39 4. 2006). 2008.205 d-1. The specific growth rate was higher. . cruentum respectively (Sobczuk et al. temperature and pH were continuously monitored. tricornutum and P. Figure 4. not only during samples as was the case with the other experiments... as the rpm was increased during steady state so did the biomass concentration up to 350rpm and 550rpm for P. Hellebust et al. The purpose of the first experiment was to test the effect in biomass concentration when changing the mixing speed after biomass concentration had reached steady state. at a value of 0. Not much has been researched on the effect of mixing speed on microalgae but there is a study the effect of mixing speed on two strains of microalgae. Bold’s media was used for all experiments performed in the CSTR. Funahashi et al. when the culture was mixed at 350rpm than when mixed at 150rpm (µ=0.123 d-1). These results are consistent with what is reported in literature for microorganisms grown in CSTR. increase of mixing speed improves overall cell growth and productivity (AguirreEzkauriatza et al. Phaedactylum tricornutum and Porphyridium cruentum.. all values are very close except for the mixing speed and the resulting specific growth rates and maximum biomass concentration.. Fenice et al.6 and Table 4. 1973). 2012. 1999. The purpose of the second experiment was to use the best mixing speed found through the first experiment and test different flow rates. It is clear that the higher mixing speed had a positive effect on the growth rate and final biomass concentration.3 Batch Cultivation in Stirred Tank Photo-bioreactor. Both experiments were run for around 17-18 days.. 62 6. light intensity (lux) 7150 7300 Month of exp.63 3. Temperature (ºC) % CO2 Avg. Chlorella vulgaris in Bold’s media.205 Doubling time (days) 5.470 0.40 Table 4. (g/l) 0. Biomass Conc.590 Avg.39 Max. CO2 conc. Aug Oct Working Volume (L) 10 10 Inoculum Concentration (%) 20 20 Additional Light Source Yes Yes 3 3 235 148 2 2 Specific Growth Rate.123 0. Experiment (rpm) CSTR1 (150) CSTR2 (350) 25 25 Avg. in culture (ppm) Air Flow Rate (L/min) . µ (d-1) 0.2: Parameters and results of CSTR experiments. pH 6.61 Avg. 1 0.6: Effect of mixing speed on Chlorella vulgaris cultivated in a CSTR Since the flowrate of air remains constant within the two experiments.7 Biomass Concentration (g/L) 0. the positive changes in biomass concentration are due to the effects of a higher mixing speed which led to the CO2 in the air being more readily available to microalgae.41 0. .4 0. 2012).0 0 5 10 15 Days Note: Error bars represent standard deviation from the average biomass concentration in steady state conditions Figure 4.3 0. which increases the concentration of CO2 in the media therefore making CO2 more readily available to cells in the culture (Kazim.6 0.7 it can be seen that at 300 rpm air bubbles are broken up into a smaller size bubbles than the ones observed when mixing at 150 rpm.2 150 rpm 350rpm 0.5 0. In Figure 4. they tested various . 2005) found a similar result..177 g/L. Both of these experiments had an additional source of carbon.32 d-1 but with a higher final biomass concentration of 0. Off all the media formulations.345 d-1 but the maximum biomass concentration achieved was only 0. parameters and results can be compared.4 Batch Cultivation in Tubular and Continuously Stirred Tank Photo-bioreactors When the results of the CSTR experiments performed are placed side by side (Table 4. A study by (Muñoz et al. 2012) 4. A similar µ value was achieved on experiment B3 at 0. Bold’s media with added NaHCO3 showed the highest specific growth rate (µ) of all the experiments at 0. Both of the CSTR experiments presented no better growth rates or maximum biomass concentrations than the other experiments even when they were bubbled with 3% CO2 enriched air.445 g/L.7: Turbulence inside the stirred tank reactor at different mixing (Kazim. one in the form of carbonate (NaHCO3) and the other in the form of gaseous CO2 mixed in with the bubbled air.3) with those obtained in the two other configurations. 4L flask and tubular photobioreactor.42 150 rpm 300 rpm Note: Air flowrate = 2 L/min Figure 4. and they had the highest specific growth rates of all the experiments but not the highest biomass concentration. 62 6.470 0. Sep Jun Aug Oct Working Volume (L) 6.01 5.63 3.0 23.445 0.590 .123 0.43 microalgae species. including Chlorella vulgaris and Chlorella sorokiniana. light intensity (lux) 860 8500 7150 7300 Month of exp. for the removal of the carcinogenic acetronitrile from wastewater digester sludge on both tubular and stirred tank reactors. Experiment Number/Media (RPM) B3 NaHCO3 CSTR1 (150) CSTR2 (350) Avg.3: Comparison of Batch Studies with Chlorella vulgaris on different photobioreactor configurations. Table 4. The highest acetonitrile removal rates and biomass density were shown by Chlorella sorokiniana when cultured in a tubular photo-bioreactor.5 10 10 Inoculum Concentration (%) 30 15 20 20 Additional Light Source Yes No Yes Yes % CO2 2 No 3 3 Air Flow Rate (L/min) 2 --- 2 2 Specific Growth Rate.205 Doubling time (days) 2.0 3.54 9.4 25 25 Avg. Biomass Conc. pH 6. (g/l) 0. µ (d-1) 0.345 0.32 0.39 Max. Temperature (ºC) 21.61 Avg.177 0.80 6.17 2. a decrease of biomass concentration (beginning washout of cells) was observed.or PO4-3) Biomass Concentration (g/L) 0.9 and Table 4.5 Semi-Continuous Cultivation in a Tubular Photobioreactor For the continuous part of the experiment in the tubular photo-bioreactor (PBR). Biomass concentration steadily increased with the dilution rate (3).7 d-1 -1 400 0.4 summarize the effect that different dilution rates had on the maximum biomass concentration achieved by Chlorella vulgaris when it reached steady state.44 4. temperature and pH were measured.6 250 Biomass Nitrate Phosphate 0. At a dilution rate of 0.0 0 20 40 60 Days Figure 4.5 d-1 350 0. it stayed constant through the rest of the experiment.2 50 0 0.8: Semi-continuous cultivation and nutrient consumption of C.40 respectively.75 d-1. Figure 4. pH was controlled and kept in range with a 1N HCl solution.6 d-1 300 0. The concentration of nitrates decreased rapidly within the first 20 days to the point of almost being depleted. the average was 23°C and 7.0 0. vulgaris in a tubular photo-bioreactor.8 0. Phosphates in the media were used sparingly and the concentration remained constant . 450 1.75 d 100 0.4 200 150 mg/L (NO3 . 80 Dilution Rate (day-1) Note: Error bars represent standard deviation from the average biomass concentration in steady state conditions Figure 4.65 0. which was slightly higher than the maximum concentration reached during the batch phase which was 0. which equals the dilution rate (3) in steady state therefore µ=0..79 g/L at 3=0.55 0. Equation (2.70 0. suggested that the dissolved inorganic phosphate had no effect on the cell mass production (Su et al.50 0.60 0.9: Steady state behaviour of C.68 g/L.99 days.45 through most of the experiment.75 0. The highest biomass concentration reached through this experiment was 0.80 0.70 0. vulgaris. of the cyanobacterium Thermosynechoccus. . The results of a study. 2012).99 days.60 0.7 d-1 in this experiment which gives a doubling time (td) is 0.55 0. Average Biomass Concentration (g/L) 0.85 0. this means that the amount of microalgae in the culture will double every 0. Nitrates were reduced by almost 100% and phosphates only by 67%.7 d-1.75 0.50 0.45 0.65 0.10) can be used to find the specific growth rate (µ). 75 0. Results are shown on Figure 4.79 0.1 Effect of Agitation Speed on Cell Growth A stirred tank reactor was used to test the effect of mixing speed on the biomass concentration of Chlorella vulgaris. a dilution rate of 0. once the culture has passed the exponential growth phase and had reached steady state.6 Dilution Rate.7 0. the run was started at a mixing speed of 150 rpm. steady state was reached again and speed was increased to 450 rpm.73 0. Biomass concentration increased steadily along with the increase of mixing speed. . It can also be observed that the concentration of CO2 decreased within the first 20 days of the experiment until steady state was reached at 150 rpm and then remained constant through the rest of the experiment.46 Table 4.5 0. Table 4.6.10.67 0.6 0.54 CSTR 4.5 shows the maximum biomass concentration reached with the different mixing speeds.4: Steady state biomass concentration at different dilution rates in a tubular photo-bioreactor 4.4 d-1 was set and the speed was increased to 250 rpm until the culture reached steady state again then the mixing speed was increased to 350 rpm. 7 Biomass Concentration (d-1) (g/L) 0. 1992).8 300 100 50 20 0. At a constant air flow rate. . Dilution rate.4 d-1 Figure 4. They found that the biomass concentration increased with agitation speed up to 350rpm. 0. in the case of Phaeodactylum tricornotum. an increase in agitation speed (rpm) can cause cell damage this can be mostly due to the air bubbles in the medium undergoing breakup and coalescence in the impeller stream region that encompasses spinning impellers (Yang and Wang. and up to 550rpm in the case of Porphyridium cruentum and found drastic decrease in biomass concentration at higher speeds..4 250 160 mg/L (NO or PO4 ) Biomass Concentration (g/L) 350 150 ppm CO2 450 0.0 0 0 10 20 30 40 0 50 Days Note: numbers on squares is the mixing speed in rpm.47 200 180 120 100 150 80 Biomass Nitrate Phosphate CO2 0. (Sobczuk et al.6 0. 2006) measured damage to microalgae cells by quantifying the decrease of biomass concentration after steady state had been achieved at the mixing speeds tested.2 60 40 -3 140 200 -1 250 0.10: Effect of Mixing Speed on Cell Growth and Biomass Concentration It was expected that at higher mixing speeds there would be visible cell damage but none was observed through the microscope at the maximum mixing speed tested of 450rpm. 5: Biomass concentration at different mixing speeds in a CSTR 4. the mixing speed was fixed at 350 rpm and changed the dilution rate of the system once it reached steady state. As 3 values increased and the culture reached steady state. 2006) but as mixing speed was increased to 450rpm.6 d-1 and 0.5 d-1 was started. Nitrates were reduced by 80% and phosphates by 20% within the batch phase of the experiment. .75 d-1. where a decrease of biomass concentration in the bioreactor due to the washout of the cells was noticed.48 In this experiment. in which the effect of mixing speed on biomass concentration of Chlorella vulgaris was tested.11. Three dilution rates (3) were tested in this experiment. Table 4.. going only from 0.656 Effect of Dilution Rate on Cell Growth In order to investigate the effect of different dilution rates in the growth of Chlorella vulgaris.637 g/L at the previous speed of 350rpm. to 0.2 Mixing speed Biomass Concentration (rpm) (g/L) 150 0. 0. the concentration of both nitrates and phosphates increased slightly as was expected.6. biomass concentration values were higher up until a dilution rate of 0. which lasted about 14 days. but concentration of both nutrients reached a steady value that remained through the rest of the experiment.467 250 0. The mixing speed of 350rpm was chosen for further experiments to save on energy and to avoid possible cell damage due to long periods of high speed mixing at 450rpm.637 450 0. it was found to have the same initial effect as the study done by (Sobczuk et al.5 d-1. The results are illustrated in Figure 4.656 g/L at 450rpm and no visible cell damage was noticed. When the first dilution rate of 0. 0. it was found that it had little effect in the increase of biomass. since fresh media was being added to the culture in the bioreactor.75 d-1.574 350 0. 5 d 0. this means that the amount of microalgae in the culture will double every 1. The highest biomass concentration reached was 0.15 days.15 days.2 120 20 0. 220 1. in steady state 3=µ therefore the dilution rate at which the highest biomass concentration µ=0.10) can be used to find the specific growth rate (µ).0 -1 0.6 d-1. .8 140 40 0. which was 35% higher than the maximum concentration reached during the batch phase. as in steady state.6 d-1 200 140 -1 0.0 0 0 10 20 30 40 100 50 Days Figure 4. vulgaris in steady state.12 and Table 4. When the microalgae cell concentration is constant.6 show more clearly the effect that different dilution rates have on the maximum biomass concentration achieved by C.6 d-1 is found.49 The concentration of CO2 in the culture increased the most during the batch phase of the experiment.8 g/L at 3=0. The change in dilution rate seemed to have an effect on the concentration of CO2 which increased slightly with the increasing change in dilution rate.11: Effect of dilution rate on biomass concentration of Chlorella vulgaris in a CSTR Figure 4.6 100 80 Biomass Nitrate Phosphate CO2 0.75 d 160 0.4 60 180 160 ppm CO2 120 mg/L (NO-1 or PO4-3) Biomass Concentration (g/L) 0. equation (2. Biomass concentration steadily increased with the dilution rate (3). will have a doubling time of the cells (td) of 1. 60 0.5 0. 7 Biomass Concentration -1 (d ) (g/L) 0.6: Biomass concentration at different dilution rates in a CSTR Dilution Rate.75 0.7 0.69 0.6 .80 0.75 0.85 0.12: Biomass concentration vs dilution rate in a CSTR Table 4.4 0.5 0.6 0.50 Average Biomass Concentration (g/L) 0.65 0.8 0.3 0.8 Dilution Rate (d-1) Note: Error bars represent standard deviation from the average biomass concentration in steady state conditions Figure 4.55 0.6 0.4 0.70 0.64 0. Continuous culture of microalgae allows for maintenance of steady state over long periods of time. For most applications in engineering. It could be argued that the . The results of the continuous culture in CSTR were similar. Bold`s media with a different source of carbon in the form of NaHCO3 did not have the positive overall results that were expected. it resulted in some of the highest growth rates of all the experiments performed except for the case of when C. The negative results with C30 media were most likely due to the inhibitory effect of the higher CO2 concentration used and the form in which the iron was added to the media which could have made it biologically unavailable to the algae cells.79 g/L when the dilution rate was set at 0. vulgaris was grown semicontinuously in a tubular photo-bioreactor and Bold`s media. vulgaris was grown in C30 media. it has its main advantage on allowing for constant extraction of biomass (and any compounds it might produce) from the system. At the beginning. what is of greatest importance is the steady state behavior of a system. it reached the highest biomass concentration of 0.7 d-1. The results of the different media formulations show that C30 media and Bold`s media with added NaHCO3 are not suitable for growth of the microalgae strain Chlorella vulgaris UTEX 2714. The best overall results found in this study were when growing C. the highest concentration of biomass obtained was 0.8 g/L when the dilution rate was set at 0.6 d-1. The addition of CO2 in air was beneficial to growth and final biomass concentration. The results of this study show that Chlorella vulgaris UTEX 2714 grows better in tubular photo-bioreactors than in stirred tank reactors or 4L flasks.51 Chapter 5 5 Conclusions and Recommendations The results obtained through the experiments presented in this thesis lead to a better understanding of the behavior of the microalgae Chlorella vulgaris in different photobioreactor configurations and growth conditions. the experiment with added NaHCO3 had the highest specific growth rate of all the experiments at 0.345 d-1 but the rapid increase of pH had an adverse effect on the final concentration of biomass. ). it will mean that overall it would be possible to obtain a higher volume of culture everyday and hence a higher biomass concentration than in the case of the CSTR. Bold`s media showed to be the best media . the working volume used for this study was 10L which was the same volume than the one used in a couple of tubular photo-bioreactor experiments and higher than the 3. CSTR and 4L flask results might have not been the best within all the experiments performed but both this configurations allow for easier initial investigation of parameters.52 biomass concentration is the same so there is no difference but with this result being obtained at a dilution rate of 0.5L working volume that was utilized for the 4L flask configuration. In the case of 4L flasks there is the convenience of how easy and fast this type of set-up can be assembled. 5. also. etc. The results of this study show that tubular photo-bioreactors are amongst the best designs for the mass cultivation of microalgae. This easiness of control and tracking allowed for testing the effect of mixing speed and dilution rate on the growth of Chlorella vulgaris. helping in the reduction of CO2 emissions. nutritional and medicinal supplements) and all the benefits to the environment that this brings (treating and eliminating harmful chemicals from wastewater. vulgaris. the smaller volumes of media needed allow for multiple experiments being run at the same time which saves a lot of time since microalgae grow slower than other microorganisms like bacteria and fungi. it is easy to automatically monitor and control different parameter as the system is usually connected to a computer that keeps tracks of changes in the system. CSTR can be operated at different volumes. similar studies for other microorganisms are reported in literature but few in the case for microalgae and none found for C. CSTR still has similar advantages as those of a 4L flask set-up.1 Future Work The importance of microalgae in today`s market is emphasized by the increase need for all the products that microalgae can be used to obtain (pigments.7 d-1 for a tubular photo-bioreactor. In this study there was automatic control of temperature and mixing speed and constant tracking of CO2 concentration and pH. 53 formulation amongst those tested and even though other variations of the formulation did not show the same kind of results. the added effect of other parameters such as different light/dark cycles. it gives a starting point for testing other formula variations and the effects that certain chemical compounds can have on microalgae growth. temperature. pH and nutrient media formulations can be studied. Now that the effect of dilution rate on microalgae growth in tubular photo-bioreactor and CSTR has been studied. 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Dou, "Effect of inorganic carbon source on lipid production with autotrophic Chlorella vulgaris" Shengwu Gongcheng Xuebao/Chinese Journal of Biotechnology. 27, 436-444 (2011). 63 Appendices Appendix A: Equipment and Materials (b) (a) Figure A-1: Tubular Photo-bioreactor Set-Up. (b) 3-6L no lamps . (a) 10L with lamps. 64 Figure A-2: Sampling device for the tubular photo-bioreactor Source: ca. .vwr.com Figure A-3: Improved Neubauer hemacytometer. mV = 21. to convert the mV values given by the probe into ppm of CO2.99 -20 -40 -60 1 10 100 1000 ppm CO2 Figure A-4: Calibration Curve for CO2 Probe 10000 .584*ln(ppm CO2) – 95 (A-1) ppm CO2 = " ((mV+95)/21.65 Appendix B: Calibration Curve for CO2 The calibration curve (shown in Figure A-4) for the carbon dioxide probe was created before the start of experiments in the CSTR.548) (A-2) 80 60 40 mV 20 0 y = 21. which was later converted to Equation(A-2) . Equation (A-1) was obtained.584ln(x)-95 2 R = 0. 0 Absorbance Figure A-5: Dry Weight Calibration Curve 1.99 0.66 Appendix C: Determination of Dry Weight and Biomass Concentration A calibration curve was prepared to correlate the absorbance of the culture of Chlorella vulgaris with dry weight. which was chosen due to a preliminary study showing 686nm to be the wavelength of maximum absorbance for a culture of the strain of Chlorella used (in Bold’s media).2 0.D.25 0.20 0.30 Biomass Concentration (g/L) 0.2936*O.00 0.4 0. Equation (A-3) was obtained.0007 R2 = 0.10 y = 0.6 0.686nm+0.15 0.8 1.0007 (A-3) 0.2 .2936x+0.05 0.0 0. Biomass (dry weight) = 0. Figure A-5 shows the correlation of dry weight (biomass concentration) with the culture’s absorbance at 686nm. 065 ?/@ 0. ° 0. ° 2 days.1255 .228 0.53 days 0.228 ?/@.67 Appendix D: Sample Calculations The following is a sample calculation for the specific growth rate () and doubling time ( ): ° ° 12 days .065 12 2 0.1255 d ln 2 ln 2 5. 0. Sc. Sinaloa.2010 UAS Doctores Jovenes Scholarship (Mexico) 2008-2011 Related Work Experience Teaching Assistant The University of Western Ontario 2008-2012 .E.68 Curriculum Vitae Name: Post-secondary Education and Degrees: Claudia Sacasa Castellanos Universidad Autónoma de Sinaloa Culiacan. 2011 – 2012 Honours and Awards: CONACyT Scholarship (Mexico) 2008 . Mexico Bachelor in Biochemical Engineering 2000 – 2005 The University of Western Ontario London. Canada M. Ontario.