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Small scale cultivation of Chlorella

Sorokiniana in shake flasks: A down size
approach to uncover phenomena at the cellular
level and metabolic pathways at various
regimes of CO2 supply

M.Tech 3rd Semester Project (MTP-1)

Anshu Dutta
Roll No. 16CH60R64
Department of Chemical Engineering,
IIT Kharagpur

Supervisors: Prof. Saikat Chakraborty, Prof. Gargi Das

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DECLARATION

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CERTIFICATE

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ACKNOWLEDGEMENTS

My sincere gratefulness goes to both of my supervisors Prof. Saikat Chakraborty and Prof.
Gargi Das, for providing excellent guidance and unbelievable support. It is because of their
constant and general interest and assistance that this project has been successful.

Specially, I would like to take this opportunity to thank Prof. Saikat Chakraborty, for guiding
me and correcting my mistakes along the way. I would also like to thank, Prof. Gargi Das for
her guidance regarding the basic principles. I would like to extend my gratitude towards Prof.
Subhabrata Ray, Department of Chemical Engineering for his support and valuable inputs. I
would like to thank the members of the Algae Biofuels Research Group at Department of
Chemical Engineering, IIT Kharagpur namely Ms. Ashvini Nair, Mr. Arun Kumar Mehta, Dr.
Shailesh S. Sawant for their valuable suggestions and helpful discussions. I would also like to
thank my family and friends who have been a source of encouragement and inspiration
throughout the duration of this project.

Finally, I would like to express my utmost gratitude to my parents, for their unconditional
affection and support.

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OBJECTIVES

The main objectives of this experimental project and study were:

Culturing the micro-algal species Chlorella Sorokiniana under different regimes of

CO2 supply (very low: 0% (v/v) CO2 supply; low: atmospheric concentration of CO2
supply; high: 1% (v/v), 3%(v/v), 5%(v/v) CO2 supply) for around 6days and observe
the biomass growth profiles, total chlorophyll content as well as biomass
productivities over time (immediate and overall) for each cases.

To study the effect of CO2 environment on elemental composition(C, H, N, S) and

macronutrient composition (Carbohydrate, Lipid and Protein).

To study the effect of 18L-6D light cycle on Night Time Biomass Loss (NTBL) and
draw inferences on overall growth and metabolic losses during dark hours under
different regimes of CO2 supply.

To develop a lumped model based on central metabolic network of Chlorella species

and infer the portioning of biomass production towards different macronutrient
composition under different regimes of CO2 supply.

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CONTENTS

TITLE Page No.

Declaration
Certificate
Acknowledgement iv
Objectives v
Contents vi-vii
List of Figures viii-xi
List of Tables xii
Chapter 1: Introduction 1-7
Chapter 2: Literature Review 8-16
Chapter 3: Materials and Methods 17-25

3.1. Materials 17

3.2. Instruments/Equipments 17-20

3.3. Experimental Procedure
20-21
3.4. Calculations and Methods of Estimation
21-25
Chapter 4: Results and Discussions 26-55

4.1. Mean Absorbance and Mean Calibrated Biomass Conc.(g/L) for 26-35
different runs
4.2. Mean Biomass Productivities [Immediate: (g/L/hr) and Overall:
36-43
(g/L/day)] for different runs (based on calibrated biomass
concentration)
4.3. Average Declivity/Productivity during night time biomass loss
44-45
and Average % Night Time Biomass Loss for different runs
4.4. Total Chlorophyll Content (Mean): (g/mL)] for different runs
4.5. CHNS Analysis DATA Elemental composition of biomass for 46-50

different runs
4.6. Macronutrient composition of biomass for different runs 51-52

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52-55
Chapter 5: Future Work 56-58

Chapter 6: Conclusions 59

References 60-65

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LIST OF FIGURES

Figure Details Page no.

Figure 1 - Transesterification of oil to biodiesel 1
Figure 2: Typical phases of the batch microorganism growth 4
Figure 3: Growth rate as a function of temperature. 4
Figure 4 : Flowchart to delineate the various types of metabolism (trophism) 6
Figure 5 - Algal cultivation system using a two phase process. 7
Figure 6: Overview of an algal cell in the light, showing the main metabolic 9
processes.
Figure 7: Schematic representation of the CBB cycle around RuBisCO 10
Figure 8: A simplified model of the cyanobacterial and microalgal CO2 12
concentrating mechanism (CCM).
Figure 9: Schematic model of the Chlamydomonas CO2 concentrating 13
mechanism (CCM) in low CO2 and very low CO2.
Figure 10: Schematic model of the Chlamydomonas CO2 concentrating 16
mechanism (CCM) in high CO2 supply.
Figure 11: Schematic Representation of Photobioreactor (PBR) Setup for 18
small scale cultivation of C.Sorokiniana used in the experimental project
Figure 12: Carbohydrate Standard Calibration Curve 24
Figure 13: Protein Standard Calibration Curve 24
Figure 14: Time profile of mean absorbance values of biomass samples at 26
0%(v/v) CO2 supply
Figure 15: Calibration curve for biomass concentration based on absorbance 26
at 0%(v/v) CO2
Figure 16: Variation of calibrated Biomass Concentration with Time at atm. 27
CO2 supply
Figure 17: Time profile of mean absorbance values of biomass samples at 27
atm. CO2 supply

Figure 18: Calibration curve for biomass concentration based on absorbance 28

at atm. CO2 supply

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Figure 19: Variation of calibrated Biomass Concentration with Time at atm. 28

CO2 supply
Figure 20: Time profile of mean absorbance values of biomass samples at 29
1%(v/v) CO2 supply
Figure 21: Calibration curve for biomass concentration based on absorbance 29
at 1%(v/v) CO2 supply
Figure 22: Variation of calibrated Biomass Concentration with Time at 30
1%(v/v) CO2 supply
Figure 23: Time profile of mean absorbance values of biomass samples at 30
3%(v/v) CO2 supply
Figure 24: Calibration curve for biomass concentration based on absorbance 31
at 3 %(v/v) CO2 supply
Figure 25: Variation of calibrated Biomass Concentration with Time at 31
3%(v/v) CO2 supply
Figure 26: Time profile of mean absorbance values of biomass samples at 32
5%(v/v) CO2 supply
Figure 27: Calibration curve for biomass concentration based on absorbance 32
at 5%(v/v) CO2 supply
Figure 28: Variation of calibrated Biomass Concentration with Time at 33
5%(v/v) CO2 supply
Figure 29: Comparison of time profiles of mean absorbance of biomass 33
samples for different runs
Figure 30: Comparison of variations of calibrated biomass concentrations 34
with time for different runs
Figure 31: Maximum Biomass Concentrations achieved at different sampling 34
times for different runs
Figure 32: Variation of Immediate productivities with Time at 0%(v/v) CO2 36
supply
Figure 33: Variation of Immediate productivities with Time at atm. CO2 36
supply
Figure 34: Variation of Immediate productivities with Time at 1%(v/v) CO2 37
supply
Figure 35: Variation of Immediate productivities with Time at 3%(v/v) CO2 37

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supply
Figure 36: Variation of Immediate productivities with Time at 5%(v/v) CO2 38
supply
Figure 37: Comparison of time profiles of immediate productivities for 38
different runs
Figure 38: Variation of Overall productivities with Time at 0%(v/v) CO2 39
supply
Figure 39: Variation of Overall productivities with Time at atm. CO2 supply 40
Figure 40: Variation of Overall productivities with Time at 1%(v/v) CO2 40
supply
Figure 41: Variation of Overall productivities with Time at 3%(v/v) CO2 41
supply
Figure 42: Variation of Overall productivities with Time at 5%(v/v) CO2 41
supply
Figure 43: Comparison of time profiles of overall productivities for different 42
runs
Figure 44: Comparison of maximum overall biomass productivities attained 42
at different sampling times for different runs
Figure 45: Comparison of Average Declivity/Productivity during Night Time 44
Biomass Loss for different runs
Figure 46: Comparison of Average % Night Time Biomass Loss for different 44
runs
Figure 47: Time profile of Total Chlorophyll content for 0%(v/v) CO2 supply 46

Figure 48: Time profile of Total Chlorophyll content for atm. CO2 supply 46
Figure 49: Time profile of Total Chlorophyll content for 1%(v/v) CO2 supply 47
Figure 50: Time profile of Total Chlorophyll content for 3%(v/v) CO2 supply 47
Figure 51: Time profile of Total Chlorophyll content for 5%(v/v) CO2 supply 48
Figure 52: Comparison of time profiles of total chlorophyll content for 48
different runs
Figure 53: Comparison of Maximum Total Chlorophyll content attained at 49
different sampling times for different runs
Figure 54: Actual Photographs of Reactor Setup and Visual comparison of 50
pigment content with different (v/v %) CO2 supply

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A: 0% B: ATM. C: 1%, 3% D: 5% E: High C02 supply (1%, 3%, 5%)

Figure 55: Comparison of Elemental composition of Biomass for different 51
runs based on CHNS analysis data
Figure 56: Comparison of % Carbohydrate content of biomass for different 52
runs
Figure 57: Comparison of % Lipid content of biomass for different runs 52
Figure 58: Comparison of maximum lipid productivities for different runs 53
Figure 59: Comparison of % Protein content of biomass for different runs 53
Figure 60: Comparison of Macronutrient Composition of Biomass for 54
different runs
Figure 61: Simplified metabolic model for Chlorella species based on 56
lumping of reaction routes

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LIST OF TABLES

Table Details Page no.

Table 1: Cellular location, putative function, family of various proteins within 16
the algal cell at different CO2 acclimation states
Table 2: C/N ratio vs. Carbs/Protein ratio at various % CO2 supply 55

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