effects of dissolved inorganic carbon, ph, and light on growth and lipid accumulation in

1

Effects of Dissolved Inorganic Carbon, pH, and Light on

Growth and Lipid Accumulation in Microalgae

A dissertation submitted to the

Division of Graduate Studies and Research

of the University of Cincinnati

in partial fulfillment of the requirements

of the degree of

Doctor of Philosophy (Ph.D.)

In the Department of Biomedical, Chemical, and Environmental Engineering

of the College of Engineering & Applied Science

2014

by

Jinsoo Kim

M.S. Chemical and Biological Engineering, Seoul National University, 2006

B.S. Chemical Engineering, Chungnam National University, 2003

Committee: Dr. Joo-Youp Lee (Chair)

Dr. Junhang Dong

Dr. Soon-Jai Khang

Dr. Timothy C. Keener

Dr. Carole Dabney-Smith

i

ABSTRACT

A primary objective of this study is to investigate the feasibility of using sodium

bicarbonate (NaHCO3) as a buffer to increase dissolved inorganic carbon (DIC) concentrations in

a culture medium for the growth of microalgae and the effects of DIC concentrations, pH and

light on growth and lipid accumulation in microalgae. Another objective is to investigate the

feasibility of removing residual nutrients such as nitrogen and phosphorus from wastewater using

microalgae. C. vulgaris was used to remove residual NH3/NH4+ and PO4

3- from secondary

wastewater effluent. C. vulgaris could effectively remove nitrogen and phosphorus under

autotrophic growth, and the removal rate could be promoted by a high initial biomass

concentration (e.g. ~350 mg/L). A Monod model was used to express the growth kinetics with a

limiting substrate.

This study has found that NaHCO3 can play a critical role as an excellent buffer that can

keep the DIC concentration high within an appropriate pH range for the growth of microalgae.

The use of high DIC concentrations can significantly increase the growth rate of C. vulgaris and

N. oleoabundans under a nutrient-sufficient condition. However, a sodium (Na+) ion

concentration should not exceed ~60 mM for C. vulgaris and ~100 mM for N. oleoabundans

because the high salinity derived from Na+

might limit their growths.

A high DIC concentration did not significantly impact on the lipid accumulation in N.

oleoabundans under a nutrient-sufficient condition. However, under nitrogen deprivation, a high

DIC concentration could help significantly increase neutral lipid accumulation in N.

oleoabundans. In addition, pH control was found to play an important role in total lipid

accumulation in N. oleoabundans under nitrogen deprivation condition.

ii

The growth of C. vulgaris increased with an increase in incident light intensity (Iin).

However, the specific growth rate with respect to average light intensity ( I ) decreased when Iin

increased (photoinhibition effect). The overall specific growth rate significantly decreased due

to an increase in biomass concentration (photolimitation). Based the above observations, a

model for the growth of C. vulgaris under a nutrient-sufficient condition was constructed by

taking into account the specific growth and average light intensity. The model could predict the

growth of C. vulgaris with a reasonably good accuracy in terms of incident light intensity and

reactor size.

iii

Copyright © 2014 by Jinsoo Kim

All rights reserved

iv

ACKNOWLEDGEMENTS

I would like to express my great appreciation for my advisor, Dr. Joo-Youp Lee for his advice,

encouragement, inspiration, guidance and financial support. I would like to acknowledge the

MSD and NSF REU programs for providing financial support for my Ph.D. degree. I would like

to appreciate the consistent efforts of my committee members, Dr. Soon-Jai Khang, Dr. Junhang

Dong, Dr. Timothy C. Keener, and Dr. Carole Dabney-Smith who are willing to provide

valuable suggestions with their knowledge and expertise so that I can successfully complete my

dissertation. I would like to thank Ms. Badkas Apurva for helping with revisions of my

dissertation. I would like to sincerely appreciate to my family, Ms. Ok-Ja Jeon, Mr. Chung-Sung

Kim, and Ms. Sun-Eun Kim for their physical, financial and mental assistance. Finally, I would

like to express great thanks to God for guiding and keeping my life safe, giving me peace and

power whenever I am facing difficulties.

I

Table of Contents

Nomenclatures ....................................................................................................... VI

List of Acronyms and Abbreviations ............................................................... VIII

List of Figures .......................................................................................................... X

List of Tables ...................................................................................................... XIII

Chapter 1. Introduction ........................................................................................ 1

1.1. The Importance of CO2 Reduction and Microalgae for CO2 Control .............................. 1

1.2. Problem Statement of Current Technology for the Cultivation of Microalgae ................ 1

1.3. Objective of This Research ........................................................................................... 3

1.4. References ........................................................................................................................ 3

Removal of Nitrogen and Phosphorus from Municipal Wastewater Chapter 2

Effluent using Chlorella vulgaris and Its Growth Kinetics .................................. 6

2.1. Introduction ...................................................................................................................... 6

2.2. Materials and Methods ..................................................................................................... 7

2.2.1. Cultivation Medium and Conditions ..................................................................... 7

2.2.2. Determination of Cell Density of C. vulgaris ....................................................... 8

2.2.3. Determination of TIC Concentration .................................................................... 8

2.2.4. Determination of PO43-

and NH3/NH4+ Concentrations ........................................ 9

2.3. Results and Discussion ..................................................................................................... 9

2.3.1. Growth and Nutrients Removal with Low Initial Cell Density ............................ 9

2.3.2. Growth and Nutrients Removal with High Initial Cell Density .......................... 13

II

2.3.3. Growth Kinetics of C. vulgaris ........................................................................... 14

2.4. Conclusions .................................................................................................................... 17

2.5. Acknowledgement .......................................................................................................... 17

2.6. References ...................................................................................................................... 18

Growth of Chlorella Vulgaris using Sodium Bicarbonate under No Chapter 3

Mixing Condition ...................................................................................................21

3.1. Introduction .................................................................................................................... 21

3.2. Material and Methods..................................................................................................... 22

3.2.1. Culture Medium and Conditions ......................................................................... 22

3.2.2. Determination of Cell Mass Density of C. vulgaris ............................................ 23

3.2.3. Determination of DIC Concentration .................................................................. 24

3.2.4. Analytical Methods ............................................................................................. 25

3.3. Results and Discussion ................................................................................................... 25

3.3.1. Use of Sodium Bicarbonate for Increasing DIC Concentrations ........................ 25

3.3.2. Growth of C. vulgaris in NaHCO3 Solutions ...................................................... 27

3.3.3. External Mass Transfer of DIC from Bulk culture Medium to Cell Surface ...... 29

3.4. Conclusions .................................................................................................................... 32


3.6. References ...................................................................................................................... 33

Effects of Dissolved Inorganic Carbon and Mixing on Autotrophic Chapter 4

Growth of Chlorella vulgaris .................................................................................37

4.1. Introduction .................................................................................................................... 37

4.2. Materials and Methods ................................................................................................... 38

III

4.2.1. Culture Media and Conditions ............................................................................ 38

4.2.2. Determination of Cell mass and Number Densities of C. vulgaris ..................... 39

4.2.3. Determination of DIC, NH3/NH4+, and PO4

3- Concentrations ............................ 40

4.2.4. Determination of Carbon Content in C. vulgaris ................................................ 40


4.3.1 Comparison of Equilibrium DIC Concentrations with and without NaHCO3 ........ 40

4.3.2. Growth of C. vulgaris under Different DIC Concentrations and Mixing

Conditions ........................................................................................................................... 43

4.3.3. External Mass Transfer of DIC from Bulk Medium to Cell Surface .................. 45

4.3.4. Effects of DIC Concentration at Cell Surface on DIC Uptake Rate of C. vulgaris

52

4.3.5. Effects of NaHCO3 Concentration on Growth of C. vulgaris ............................. 53

4.4. Conclusions .................................................................................................................... 54


4.6. References ...................................................................................................................... 55

Growth Modeling of Chlorella vulgaris with regard to Chapter 5

Photolimitation and Photoinhibition effects ........................................................60

5.1. Introduction .................................................................................................................... 60


5.2.1. Organism and Cultivation Conditions ................................................................. 61

5.2.2. Analytical Methods ............................................................................................. 62

5.2.3. Model Description ............................................................................................... 62


IV

5.3.1. Growth of C. vulgaris in Different Light Intensities on the Surface of Reactor . 65

5.3.2. Light Distribution Model with respect to Biomass Concentration ..................... 66

5.3.3. Specific Growth Rate with respect to Light Intensity ......................................... 67

5.3.4. Growth Model and Simulation ............................................................................ 69

5.3.5. Experimental Data vs. Growth Modeling ........................................................... 70

5.4. Conclusions .................................................................................................................... 71

5.5. Acknowledgements ........................................................................................................ 72

5.6. References ...................................................................................................................... 72

Effects of Dissolved Inorganic Carbon Concentrations, pH and Chapter 6

Harvest Time on Lipid Accumulation of Green Microalga Neochloris

oleoabundans in Two-stage Cultivation System ..................................................76

6.1. Introduction .................................................................................................................... 76


6.2.1. Culture Medium and Conditions ......................................................................... 78

6.2.2. Determination of Cell Density of N. oleoabundans ............................................ 80

6.2.3. Determination of DIC, NO32-

, and PO43-

Concentration ..................................... 80

6.2.4. Determination of Total Lipids by Gravimetric Method ...................................... 81


6.3.1. The first-Stage Phase: Growth of N. oleoabundans under Different DIC

Concentration under Nutrient-Sufficient Condition ............................................................ 83

6.3.2. First-Stage Phase: Lipid Accumulation in N. oleoabundans under Different DIC

Concentration for the First-Stage Phase .............................................................................. 84

V

6.3.3. Second-Stage Phase: Growth of N. oleoabundans under Different DIC

Concentration under Nitrogen-Deprivation Condition ....................................................... 87

6.3.4. Second-Stage Phase: Lipid Accumulation in N. oleoabundans under Different DIC

Concentration under Nitrogen-Deprivation Condition ....................................................... 90

6.4. Conclusions .................................................................................................................... 91


6.6 References ...................................................................................................................... 92

Summary and Suggested Future Studies ..........................................96 Chapter 7

7.1. Summary ........................................................................................................................ 96

7.2. Suggested future studies ................................................................................................. 99

VI

Nomenclatures

A(t) Total surface area (m2)

α Absorption coefficient (m2∙g

-1)

CB(t) Bulk DIC concentration (mol/m3)

CS(t) Surface concentration of DIC (mol/m3)

D Length of a magnetic stirrer in meter (m)

DDIC,water Diffusivity value for DIC at 25 C

dp Diameter of an algal cell (3-µm)

Iin Light intensity (µmol∙m-2

∙s-1

)

I Average light intensity (µmol∙m-2

∙s-1

)

kI Saturation concentration (µmol∙m-2

∙s-1

)

kc Liquid-solid mass transfer coefficient (m/s)

Ka Absorption coefficient (m2/g)

Ks Monod coefficient (mg/L)

n Fitting parameter

N Impeller speed (rpm)

Pe Peclet number

R Reactor radius (m)

r0 Light path (m)

cr Radius of the force vortex (m)

Rep Reynolds number

S Concentration of a limiting nutrient (mg/L)

Sc Schmidt number

Sh Sherwood number

VII

t Time (h)

θ Angle of the light path (radian)

δ Film thickness (m)

Kinematic viscosity of water (m2/s)

Vl Liquid volume (m3)

µ Specific growth rate (h-1

)

µmax Maximum growth rate coefficient (h-1

)

u Tangential velocity (m/s)

( )u r Average velocity of the algae cell moving in the direction (m/s)

W Biomass concentration (mg/L)

VIII

List of Acronyms and Abbreviations

CA Carbonic anhydrase enzyme

(Ca(H2PO4)2·H2O) Calcium phosphate monobasic monohydrate

CO2 Carbon dioxide

CO32-

Carbonate ion

C. vulgaris Chlorella vulgaris

CuSO4·5H2O Cupric sulfate

DIC Dissolved inorganic carbon

FeCl3 Iron chloride

H+ Hydrogen ion

HCl Hydrochloric acid

HCO3- Bicarbonate ion

H2CO3 Carbonic acid

H3BO3 Boric acid

KCl Potassium chloride

(MgSO4·7H2O) Magnesium sulfate hetahydrate

MnCl2·4H2O Manganese chloride

NaHCO3 Sodium bicarbonate

Na2MO4·2H2O Sodium molybdate

N. oleoabundans Neochloris oleoabundans

NH3/NH4+ Ammonia/ammonium ion

((NH4)2SO4) Ammonium sulfate

OH- Hydroxyl ion

PO43-

Orthophosphate ion

TIC Total inorganic carbon

IX

ZnSO4·7H2O Zinc sulfate

X

List of Figures

Figure 2.1 (a) Growth of C. vulgaris and TIC uptake and (b) TIC uptake and pH increase with

low initial algal cell density (44.37±0.60 mg/L). .......................................................................... 11

Figure 2.2 Removal of (a) nitrogen in the form of NH3/NH4+ and (b) phosphorus in the form of

orthophosphate (PO43-

).................................................................................................................. 12

Figure 2.3 (a) Growth of C. vulgaris and (b) removal of NH3/NH4+ and PO4

3- with high initial

algal cell density (354.48±2.14 mg/L). ......................................................................................... 13

Figure 2.4 (a) Determination of μmax (h-1

) and Ks (mg/L) of the Monod equation from the

Lineweaver-Burk plot of 1/μ vs. 1/S and (b) Monod plot (μ vs. S) (Substrate=nitrogen) for low

initial algal cell density. ................................................................................................................ 15



Lineweaver-Burk plot of 1/μ vs. 1/S and (b) Monod plot (μ vs. S) (Substrate=orthophosphate) for

high initial algal cell density. ........................................................................................................ 16

Figure 3.1 Growth of C. vulgaris in different initial DIC concentrations. ................................... 27

Figure 3.2 Temporal DIC concentration profiles in culture media with different initial DIC

concentrations. .............................................................................................................................. 29

Figure 3.3 (a) Temporal DIC concentration profile at the cell surface in culture media with

different initial DIC concentrations (b) Comparison of calculated bulk DIC concentrations with

measured bulk DIC concentrations, and (c) Diffusive flux of DIC. ............................................. 32

Figure 4.1 Effects of different DIC concentrations and mixing conditions on the growth of C.

vulgaris: (a) initial DIC of 15 mg C/L under different mixing speeds; (b) initial DIC of 144 mg

C/L under different mixing speeds; (c) pH change under different DIC and mixing speeds. Note:

CO2(g) was added to all culture media to control the pH of the media at 7 once daily; (d)

XI

nitrogen concentration at 192 hours under different DIC concentrations and mixing speeds; (e)

phosphorus concentration at 192 hours under different DIC concentrations and mixing speeds. 43

Figure 4.2 A schematic of rotational motion generated by a magnetic stirrer in a batch reactor. 48

Figure 4.3 Comparison of bulk DIC concentrations (CB) with surface DIC concentration (CS) in

terms of different initial DIC concentrations and mixing speeds at 192 hours. ........................... 51

Figure 4.4 Specific DIC uptake rate of C. vulgaris with respect to different DIC concentrations

(15, 30, 144, and 712 mg C/L) at the cell surface. ........................................................................ 52

Figure 4.5 Effect of high concentrations of DIC (146, 718, and 1431 mg C/L) and mixing (0 and

125 rpm) on the growth of C. vulgaris. ........................................................................................ 54

Figure 5.1 (a) Growth of C. vulgaris with respect to time dependent on light intensity (0, 30, 55,

80, 197, 476, and 848 µmol∙m-2

∙s-1

) on the surface of reactor, (b) Specific growth rate (h-1

) with

respect to time dependent on light intensity, and (c) Specific growth rate (h-1

) with respect to

biomass concentration. .................................................................................................................. 65

Figure 5.2 (a) Schematic representation of light distribution model, and (b) simulation vs.

experimental data of the light distribution with respect to the r0 inside reactor. .......................... 67

Figure 5.3 Specific growth rate with respect to average light intensity depending on light

intensity on the surface of reactor. ................................................................................................ 68

Figure 5.4 (a) Simulated a specific growth rate with respect to time, (b) light intensity with

respect to the light path and time, (c) average light intensity with respect to time, and (d) growth

of C. vulgaris with respect to time. ............................................................................................... 69

Figure 5.5 Comparison between experimental data and simulated data (a) growth kinetics of C.

vulgaris with respect to time depending on light intensity on the surface of reactor, and (b)

growth kinetics of C. vulgaris with respect to time depending on the reactor size. ..................... 70

XII

Figure 6.1 Effects of different DIC concentrations on the growth of N. oleoabundans under

nutrient-sufficient condition during first-stage phase: (a) initial DIC of 0.001 M, 0.003 M, 0.005

M, 0.01 M, 0.03 M, 0.05 M, 0.1 M, 0.3 M, and 0.5 M of NaHCO3/L under 350 rpm mixing

speeds. Note: CO2(g) was added all culture media to control the pH of the media at 7 once daily;

(b) nitrogen concentration at 312 h under different DIC concentrations; (c) phosphorus

concentration at 312 h under different DIC concentrations; (d) pH change under different DIC

concentrations. .............................................................................................................................. 82

Figure 6.2 Neutral and polar lipids content in N. oleoabundans determined by gravimetric

method in different DIC concentrations under nutrient-sufficient condition. .............................. 85

Figure 6.3 Effects of different DIC concentrations, existence of pH control, and harvest time on

the growth of N. oleoabundans under nitrogen-deprivation condition during second-stage phase:

(a) initial DIC of 0.003 M and 0.01 M of NaHCO3/L under 350 rpm mixing speeds. Note: CO2(g)

was added S5, S6, S9, and S10 culture media to control the pH of the media at 7 once daily; (b)

pH change in different DIC concentrations, existence of pH control. .......................................... 87


method in different DIC concentrations under nitrogen-deprivation condition during second-

stage phase. ................................................................................................................................... 90

XIII

List of Tables

Table 2.1 Characteristics of wastewater samples in Mill Creek plant .......................................... 10

Table 3.1 Dissolved Inorganic Carbon (DIC) concentrations generated from CO2(g) absorption

and sodium bicarbonate dissolution at 1 atm. ............................................................................... 26

Table 3.2 Coefficients of a third-order polynomial function in Eq. (11) for total surface area. ... 30

Table 3.3. Coefficients used for surface DIC concentrations in Eq. (13). .................................... 31

Table 4.1 DIC concentrations generated from CO2(g) absorption and NaHCO3 dissolution at 1

atm................................................................................................................................................. 42

Table 4.2 Coefficients used for total surface area, A(t), and DIC surface concentrations, CS(t). 45

Table 4.3 Impeller Reynolds, flow regime, average velocity, Reynolds number, Schmidt number,

Peclet number, Sherwood number, mass transfer coefficient, and film thickness in terms of

mixing speed. ................................................................................................................................ 50

Table 5.1 Parameters of specific growth rate dependent on the light intensity on the surface of

reactor. .......................................................................................................................................... 68

1

Chapter 1. Introduction

1.1. The Importance of CO2 Reduction and Microalgae for CO2 Control

CO2 is a major contributor towards global climate change, which is estimated to be more

than 75% according to the EIA 2008 report. Most of the energy needs worldwide rely on the

combustion of fossil fuels and CO2 is the byproduct of that combustion [1, 2]. One-third of the

CO2 emissions in the USA are produced from coal-fired power plants. CO2 emission for plants

in the USA increased approximately by 18% from 1990 to 2003 and it is estimated that the

increase will go up to 54% by 2030 if CO2 emissions control is not employed [3-6]. Figueroa

estimated that approximately 33% of CO2 emissions from the plants can be reduced by using

current CO2 capture and separation techniques [5, 7].

Plants can utilize CO2 as an inorganic carbon source and can produce valuable products

such as carbohydrates and lipids. These products can be used for producing fuels such as ethanol

and biodiesel. Recently, interest in microalgae has been increasing as a feedstock for the

production of biodiesel due to their faster photosynthesis rate than terrestrial plants derived from

their high efficiency of solar energy utilization (up to 10%) [8-13]. In addition, the microalgae

are a non-edible source, producing a large amount of lipids (approximately 15-50% dry cell

weight), which grows in freshwater, brackish water, ocean and even wastewater [14]. Therefore,

it is noted that microalgae can effectively recycle CO2 to a valuable energy source.

1.2. Problem Statement of Current Technology for the Cultivation of Microalgae

Microalgae can utilize both inorganic carbon and organic carbon sources for growth.

When inorganic carbon is used for the growth of microalgae, the method is referred to as

autotrophic and when the organic carbon source is used for the growth of microalgae, the method

2

is referred to as heterotrophic growth or fermentation. Both methods can produce a large amount

of lipids but autotrophic growth can reduce the CO2 emissions. Hence, the autotrophic growth

method in this study is used for cultivation of microalgae.

CO2 concentration in water phase under atmospheric pressure is small because the

solubility of CO2(g) in water is very low under atmospheric pressure based on Henry’s law.

Hence, closed systems are generally used to increase the solubility of CO2 by increased pCO2 in

the atmosphere. However, the pH of the culture medium is decreased when the pCO2 is

increased as in the following reaction [15, 16].

2 2 3H O CO H HCO (1)

Powel et al. reported that the growth rate of microalgae is increased when inlet CO2

concentrations is increased in closed systems but the growth rate was significantly reduced when

the inlet CO2 concentration went above 20 % (v/v) [17]. Similarly, Kim et al. reported that the

autotrophic growth of microalgae was decreased at pCO2=100% because the pH of the culture

medium reached to a low pH and the acidic pH environment constituted adverse condition for

the growth of microalgae [18]. It was reported that some of the green microalgae such as C.

vulgaris, Chlorealla sp. and Dunaliella sp. were growing well in the pH window of 7-8.3 [15, 16,

19]. Hence, there is a need to study methods of increasing CO2 mass transfer while controlling

the pH of culture mediums that are agreeable to the growth of microalgae. In addition, the cost

for such closed systems are approximately four times higher than the cost of open systems and

the scale up of closed systems is difficult for mass production [20]. Therefore, it is required to

find novel method to increase CO2 concentration in water while maintaining proper pH range

under open systems.

3

1.3. Objective of This Research

The objective of this study is to increase the growth rate and lipid accumulation in

microalgae and the specific objectives are to:

(1) Investigate the ability of the microalgae to remove nitrogen and phosphorus from wastewater

effluent,

(2) Investigate the feasibility of NaHCO3 to use for the cultivation of microalgae,

(3) Investigate the effect of DIC and mixing on the growth of microalgae,

(4) Investigate the effect and establish a model of light intensity for the growth of microalgae

under a nutrient-sufficient condition, and

(5) Investigate the effect of DIC and pH for the lipid accumulation in microalgae under a

nitrogen deprivation condition.

1.4. References

[1] EIA, Greenhous Gases, Climate Change, and Energy, in, 2008.

[2] J. Skea, Slowing down global warming - a worldwide strategy - flavin,c, Energy Policy, 19

(1991) 189-191.

[3] EIA, Emissions of Greenhouse Gases in the United States 2005, in, 2006c.

[4] EIA, International Energy Outlook 2006., in, 2006b.

[5] EIA, Annual Energy Outlook 2006, in, 2006a.

[6] D.M. D'Alessandro, B. Smit, J.R. Long, Carbon Dioxide Capture: Prospects for New

Materials, Angew. Chem.-Int. Edit., 49 (2010) 6058-6082.

4

[7] J.D. Figueroa, T. Fout, S. Plasynski, H. McIlvried, R.D. Srivastava, Advancesn in CO2

capture technology - The US Department of Energy's Carbon Sequestration Program,

International Journal of Greenhouse Gas Control, 2 (2008) 9-20.

[8] Y. Chisti, Biodiesel from microalgae., Biotechnology Advances, 25 (2007).

[9] Q. Hu, M. Sommerfeld, E. Jarvis, M. Ghirardi, M. Posewitz, M. Seibert, A. Darzins,

Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and

advances., Plant Journal, 54 (2008) 621-639.

[10] T. Burton, H. Lyons, Y. Lerat, M. Stanley, M.B. Rasmussen, A review of the potential of

marine algae as a source of biofuel in Ireland, in, Dublin: Sustainable Energy Ireland-SEI,

2009.

[11] R. Davis, A. Aden, P.T. Pienkos, Techno-economic analysis of autotrophic microalgae for

fuel production, Applied Energy, 88 (2011) 3524-3531.

[12] Y. Li, D. Han, M. Sommerfeld, Q. Hu, Photosynthetic carbon partitioning and lipid

production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under

nitrogen-limited conditions, Bioresource Technology, 102 (2011) 123-129.

[13] Y.Q. Li, M. Horsman, B. Wang, N. Wu, C.Q. Lan, Effects of nitrogen sources on cell

growth and lipid accumulation of green alga Neochloris oleoabundans, Applied

Microbiology and Biotechnology, 81 (2008) 629-636.

[14] E.L. Lee, Phycology, Cambridge University Press, New York, 2008.

[15] J. Kim, J.-Y. Lee, T. Lu, Effects of dissolved inorganic carbon and mixing on autotrophic

growth of Chlorella vulgaris, Biochemical Engineering Journal, 82 (2014) 34-40.

5

[16] W. Kim, J.M. Park, G.H. Gim, S.-H. Jeong, C.M. Kang, D.-J. Kim, S.W. Kim, Optimization

of culture conditions and comparison of biomass productivity of three green algae,

Bioprocess and Biosystems Engineering 35 (2012) 19-27.

[17] E.E. Powell, M.L. Mapiour, R.W. Evitts, G.A. Hill, Growth kinetics of Chlorella vulgaris

and its use as a cathodic half cell, Bioresource Technology, 100 (2009) 269-274.

[18] A. Concas, G.A. Lutzu, M. Pisu, G. Cao, Experimental analysis and novel modeling of

semi-batch photobioreactors operated with Chlorella vulgaris and fed with 100% (v/v)

CO2, Chemical Engineering Journal, 213 (2012) 203-213.

[19] J. Kim, J.-Y. Lee, T. Keener, Growth kinetic study of Chlorella vulgaris., Topical H: Solar

Topical Conference Proceedings 2009 AICHE Annual Meeting, (2009) 8-13.

[20] L. Amer, B. Adhikari, J. Pellegrino, Technoeconomic analysis of five microalgae-to-

biofuels processes of varying complexity, Bioresource Technology, 102 (2011) 9350-

9359.

6

Removal of Nitrogen and Phosphorus from Municipal Chapter 2

Wastewater Effluent using Chlorella vulgaris and Its Growth Kinetics1

2.1. Introduction

Microalgae-derived biodiesel production has recently received great attention due to their

ability to produce triacylglycerols for biodiesel while using solar energy and CO2. A previous

life cycle assessment study showed that most energy required for autotrophic algal cultivation is

estimated to be used for the production of nutrients (e.g., nitrogen and phosphorus) and the

power consumption required to increase the mass transfer of CO2 gas into the aqueous phase

such as bubbling and mixing [1, 2]. The study recommended that ~50% of the total energy use

associated with fertilizer production could be reduced by using nitrogen and phosphorous in

wastewater. The use of wastewater for algae growth will reduce not only the cost associated

with fertilizer but also the residual nitrogen concentration present in the wastewater effluent

stream, a major contributor to ecological eutrophication [3].

The removal of nutrients such as nitrogen and phosphorus for wastewater treatment was

reported in previous studies by using Chlorella sp. and Phaeodactylum tricornutum [4-7].

Although the potential of microalgae for nutrients removal from wastewater was recognized,

little information is available on the feasibility and growth kinetics using microalgae required for

the design and operation of an algal pond. From a practical standpoint, a hydraulic retention

time required for nutrient removal is likely to be one of the major constraints for the realization

of tertiary-level wastewater treatment using microalgae [8, 9]. C. vulgaris is one of the fastest

1 Part (Introduction, Materials and Methods, results and Discussion, Conclusions and Acknowledgement) of the

content in this chapter has been published in Kim, J.; Liu, Z.; Lee, J.-Y.; Lu, T., Removal of nitrogen and phosphorus

from municipal wastewater effluent using Chlorella vulgaris and its growth kinetics. Desalination and Water

Treatment 2013, 1-7.

7

growing green microalgae and was used in this study. In this study, the feasibility of the removal

of NH3/NH4+ and PO4

3- was studied for potential tertiary wastewater treatment with different

initial cell densities. A growth model was also constructed for the design of an algal pond using

wastewater.

2.2. Materials and Methods

2.2.1. Cultivation Medium and Conditions

Secondary wastewater samples were collected from the Mill Creek plant located in

Cincinnati, Ohio, U.S.A., a major wastewater treatment facility, where ~70 and ~30% of the

wastewater is typically collected from industrial and domestic sources, respectively. The

wastewater was used as a culture medium. The capability of C. vulgaris to remove nutrients was

examined using two initial algal cell densities. For a culture with a low initial algal cell density,

a C. vulgaris sample suspended in shuisheng-4 medium was added to a 3.7-L wastewater

medium (W1) in a 4-L bottle (26 cm (height) × 15 cm (diameter)) [10]. The cell density of C.

vulgaris in the wastewater medium was found to be 44.37±0.60 mg/L after inoculation. During

the culture in the wastewater medium, the bottle was closed in order to minimize the evaporation

loss of dissolved CO2 into air, and the media were mixed by using a magnetic stirrer at a speed of

350 rpm. Then, the concentrations of NH3/NH4+, PO4

3-, and TIC (= 3HCO + 2

3CO + 32COH +

)aq(CO2 ), cell density, and the pH of the medium were measured once daily. The experiment

with a low initial algal cell density was performed in duplicate. For the culture with a high initial

algal cell density, a C. vulgaris sample concentrated by centrifugation was added to a 3.7-L

wastewater medium (W2) and the algal cell density was found to be 354.48±2.14 mg/L after

inoculation. Since the initial TIC concentration was low for the culture with a high algal cell

8

density, CO2 gas was bubbled through the medium every 12 h to supply TIC and control the pH

at ~7.

During the culture, light intensity was also kept constant. Fluorescent lamps with 6,500-

K color temperature similar to natural sunlight were used as a source of light, and the incoming

light intensity to beakers was set to 6,000 lux (100.8 µmolm-2s

-1) by controlling the distance

between the beaker and lamp. The light intensity was measured using a light intensity meter

(HQRP digital lux meter, LX1010BS, Osprey-Talon Company), and a 16-h light and 8-h dark

cycle was used for the culture.

2.2.2. Determination of Cell Density of C. vulgaris

The cell density of C. vulgaris was determined by measuring the optical density of a 15-

mL sample at 682 nm for every 24 h by using UV-vis spectrophotometer (UV-1800, Shimadzu

Scientific Instruments) [11]. The absorbance of UV spectrophotometer at 682 nm was calibrated

by measuring the weight of C. vulgaris after harvesting and drying. Then, the weight of dried

biomass was obtained from the prepared calibration curve.

2.2.3. Determination of TIC Concentration

An acid-base titration method was used to determine the concentrations of inorganic

carbon species present in the aqueous phase [12]. This titration method determines a TIC

concentration in a 15-mL sample using 0.1 N and 0.01 N HCl solutions for the titration of high

and low carbon concentrations, respectively. The accuracy of this titration method was ensured

by comparing a known amount of TIC dissolved from sodium bicarbonate or sodium carbonate

9

with the amount of TIC determined by the acid-base titration method. All TIC concentrations

were measured in duplicate.

2.2.4. Determination of PO43-

and NH3/NH4+ Concentrations

PO43-

is measured using the Phosver 3 phosphate reagent available with the HACH

Model PO-19 reactive phosphorus measurement kit. A 5-mL sample was taken from a culture

medium and a Phosver 3 phosphate reagent was added to it. After 2 min, blue color appeared

due to the presence of phosphorus, and the color intensity was proportional to the amount of an

orthophosphate concentration in the solution. The color intensity was measured using a UV-

Visible spectrophotometer (UV-1800, Shimadzu Scientific Instruments) at 890 nm.

The nitrogen concentrations in NH3 and NH4+ were measured using an ammonia probe

(Model: 9512HPBNWP Orion Thermo Scientific) [13]. All ammonium ions were converted into

ammonia by raising the pH of the sample solution of the culture medium above 12, and the

resultant ammonia concentration was determined by the ammonia probe. A 15-mL sample was

filtered out using a syringe filter (0.45 µm nominal pore with 24 mm diameter, Whatman filter)

in order to avoid potential blockage of the membrane of the ammonia probe. All the

measurements were carried out in duplicate.

2.3. Results and Discussion

2.3.1. Growth and Nutrients Removal with Low Initial Cell Density

The characteristics of the wastewater samples used for this study are summarized in

Table 2.1.

10

Table 2.1 Characteristics of wastewater samples in Mill Creek plant

Parameter

Wastewater 1 used for low initial

algal cell density experiment

(W1, mg/L)

Wastewater 2 used for high initial

algal cell density experiment

(W2, mg/L)

TIC 50.18 ± 0.83 72.24 ± 1.81

NH4+/NH3 8.05 ± 0.16 18.31 ± 0.53

PO43-

pH

1.85 ±0.10

7.34±0.05

1.37 ±0.01

7.88±0.07

The growth of C. vulgaris and uptake of TIC were measured as shown in Figure 2.1(a).

The lag-phase period required for adaptation to the wastewater condition was found to be short

(24 h), and the TIC concentration was not reduced during the period. During the growth phase,

the cell density of C. vulgaris significantly increased, and the TIC concentration sharply

decreased until 96 h, indicating active photosynthetic reaction. Microalgae can consume only

dissolved CO2(aq) and

3HCO , but, CO2(aq) concentration is much lower than

3HCO

concentration and insignificant in the pH range of 7 and 10. The carbon content in microalgae

was then estimated to be ~50% from a carbon mass balance between the inorganic carbon

consumption (36.28±1.68 mg/L) and total biomass gain (76.33±2.89 mg/L) during the entire

growth phase (96 h). The carbon content estimated from the culture of C. vulgaris is comparable

to a carbon content (~50%) of C. vulgaris reported in a previous study [2].

11

Figure 2.1 (a) Growth of C. vulgaris and TIC uptake and (b) TIC uptake and pH increase

with low initial algal cell density (44.37±0.60 mg/L).

Microalgae produce OH- when

3HCO is consumed during photosynthesis within the

algal cell by the following reaction: OHCOHCO 23 [14-16]. As a result, the pH of the

wastewater medium continued to increase from the lag phase through the growth phase as shown

in Figure 2.1(b). Then the pH started to level off as the uptake rate of TIC discontinued after 96

h. The pH of the culture medium was roughly estimated from the carbon mass balance based on

an assumption that only

3HCO ion can be consumed by C. vulgaris and OH- ion can be

generated as a result of

3HCO consumption. The estimated final pH value overpredicted the

measured pH values over the entire culture period as shown in Figure 2.1(b) probably because

some of TIC was released to the air as CO2 gas during the culture and the lost fraction was not

included in the calculation.

12

Figure 2.2 Removal of (a) nitrogen in the form of NH3/NH4+ and (b) phosphorus in the

form of orthophosphate (PO43-

).

The removal of nitrogen in NH3/NH4+ is shown in Figure 2.2(a). Overall, the nitrogen

removal showed almost the same pattern as the TIC uptake. As observed from the uptake of

TIC, a small amount (1.11±0.31 mg/L) of nitrogen concentration was consumed during the lag

phase, but then sharply decreased from 6.94±0.18 mg/L to 0.56±0.04 mg/L concomitantly with a

decrease in TIC concentration during the rapid growth phase (i.e. 24 to 96 h). The total amount

of nitrogen removed during 96 h was 7.48±0.20 mg/L. Similar to the carbon mass balance, the

nitrogen content in C. vulgaris was estimated to be ~10% from a nitrogen mass balance between

the total nitrogen removal (7.48±0.20 mg/L) and total biomass gain (76.33±2.89 mg/L) for 96 h.

The removal of PO43-

is represented in Figure 2.2(b). During the lag-phase period, a small

amount (0.08±0.02 mg/L) of phosphorus was consumed. During the growth-phase (i.e. 24 to 96

h) period, it significantly decreased from 0.47±0.03 mg/L to 0.03±0.01 mg/L. The phosphorus

content of C. vulgaris was estimated to be ~0.7% from a phosphorus mass balance between the

total phosphorus consumption (0.52±0.04 mg/L) and total biomass gain (76.33±2.89 mg/L)

13

during 96 h.

2.3.2. Growth and Nutrients Removal with High Initial Cell Density

Figure 2.3 (a) Growth of C. vulgaris and (b) removal of NH3/NH4+ and PO4

3- with high

initial algal cell density (354.48±2.14 mg/L).

The growth of C. vulgaris is shown in Figure 2.3(a) when a high (354.48±2.14 mg/L)

initial algal cell density was used for faster nitrogen and phosphorus uptake. The lag-phase

period was not observed, and PO43-

was depleted within 12 h. The concentrations of NH3/NH4+,

PO43-

, and cells were measured every 3 h until PO43-

was depleted. Almost all NH3/NH4+ were

also removed within 48 h. For this culture, the initial NH3/NH4+ concentration in this sample (i.e.

W2) was higher than that in the first W1 sample, and thus PO43-

clearly became a limiting

substrate for the growth.

14

2.3.3. Growth Kinetics of C. vulgaris

The Monod equation is a well-known substrate-limiting growth model used to describe

the growth of a microorganism covering the growth and stationary phases as shown in equation

(1):

SK

S

s max

(1)

where µmax is a maximum growth rate coefficient (h-1

), µ is a specific growth rate (h-1

), Ks is the

Monod coefficient (mg/L), and S is the concentration of a limiting nutrient (mg/L). The two

parameters, μmax and Ks, were determined from the growth and stationary phases using the

Lineweaver-Burk plot of 1/μ vs. 1/S by taking a reciprocal for each term in the Monod equation

as shown in equation (2).

maxmax

111

S

Ks (2)

For the low initial cell density case, the remaining nitrogen and phosphorus concentrations after

144 h were both low at 1.10±0.01 and 0.02±0.01 mg/L, respectively, and it was difficult to

determine a limiting substrate. Therefore, a comparison was also made for the R2 values of the

Lineweaver-Burk plot when nitrogen and phosphorus were used as limiting nutrients. The

average R2 values (from Experiments 1 and 2) for nitrogen and phosphorus were 0.97 and 0.64,

respectively. Therefore, nitrogen was determined to be a limiting substrate for the growth in the

low initial algal cell density. Then, μmax (h-1

) and Ks (mg/L) were determined to be 0.01245 (h-1

)

and 0.0696 (mg/L) for Experiment 1, and 0.01241 (h-1

) and 0.0864 (mg/L) for Experiment 2,

respectively. The experimental data were re-plotted with the Monod equation for specific

growth (μ) vs. nitrogen concentration (S) in Figure 2.4(b), and the both equations with the

determined parameters well represent the growth.

15



Lineweaver-Burk plot of 1/μ vs. 1/S and (b) Monod plot (μ vs. S) (Substrate=nitrogen) for

low initial algal cell density.

16



Lineweaver-Burk plot of 1/μ vs. 1/S and (b) Monod plot (μ vs. S)

(Substrate=orthophosphate) for high initial algal cell density.

For the culture with the high initial algal cell density, phosphorus was evidently a

limiting substrate. Following the same procedure applied above, the two parameters (μmax and Ks)

were determined to be 32.85 (h-1

) and 0.99 (mg/L). When the Monod equation was compared

for the two cases, the μ value for the culture with a high initial cell density and pH control using

CO2 gas was at least one order magnitude (e.g. ~40 times) greater than that for the culture with a

low initial cell density and no pH control. The culture with a high initial cell density and pH

control using CO2 gas could accelerate simultaneous algal cell growth and residual nitrogen and

phosphorus uptake.

In this study, the Monod expression for nutrient removal has been obtained in a batch

system. However, if an intrinsic kinetic expression is obtained, the expression can be

incorporated into a reactor mass balance model taking into account fluid flow and mass transfer

for the design of a continuous reactor. Although a possibility of nutrient uptake from wastewater

17

is demonstrated, the harvest of microalgae from a voluminous wastewater effluent stream is a

great challenge. Several options have been investigated, including chemical flocculation,

autoflocculation, filtration, flotation, sedimentation, or electrophoretic separation [17]. However,

energy-efficient, effective, and reliable harvesting methodologies for large-scale wastewater

treatment have not yet been realized and need to be developed. Total lipid content in a few

strains including Chlorella sp. and Scenedesmus sp. grown in wastewaters was reported to range

between 12−15%(wt) in dry biomass [18].

2.4. Conclusions

The growth of C. vulgaris was studied for the uptake of residual nitrogen and phosphorus

present in the secondary wastewater samples collected from a local wastewater treatment plant in

Cincinnati, OH, U.S.A. The following summary was found from this study.

1. Between nitrogen and phosphorus present in the secondary municipal wastewater

samples, either one could be a substrate limiting its growth depending on their initial

concentrations.

2. The residual nutrient removal rate can enhanced by high initial algal cell density and CO2

gas supply, and almost all nitrogen and phosphorus could be removed within 48 h.

3. The Monod equation could be used to express the algal cell growth for a limiting

substrate. It would be applicable to the design and operation of any type of continuous

photobioreactor by the incorporation into a reactor mass balance model taking into account fluid

flow and mass transfer for the removal of nutrients from wastewater.

2.5. Acknowledgement

18

This work was supported by the Metropolitan Sewer District of Greater Cincinnati

(MSDGC) under Master Services Agreement No. 85X10431, Task Order No. 0210000209. The

authors appreciate their financial support and Dr. Tim Keener for discussions.

2.6. References

[1] A.F. Clarens, E.P. Resurreccion, M.A. White, L.M. Colosi, Environmental life cycle

comparison of algae to other bioenergy feedstocks, Environmental Science and

Technology, 44 (2010) 1813-1819.

[2] L. Lardon, A. Helias, B. Sialve, J.P. Stayer, O. Bernard, Life-cycle assessment of biodiesel

production from microalgae, Environmental Science and Technology, 43 (2009) 6475-

6481.

[3] T.A. Larsen, A.C. Alder, R.I.L. Eggen, M. Maurer, J. Lienert, Source separation: will we see

aparadigm shift in wastewater handling?, Environmental Science and Technology, 43

(2009) 6121-6125.

[4] R.J. Craggs, V.J. Smith, P.J. McAuley, Waste-water nutrient removal by marine microalgae

cultured under ambient conditions in mini-ponds, Water Sci. Technol., 31 (1995) 151-160.

[5] J. Kim, B.P. Lingaraju, R. Rheaume, J.-Y. Lee, K.F. Siddiqui, Removal of ammonia from

wastewater effluent by Chlorella vulgaris, Tsinghua Science & Technology, 15

(2010) 391-396.

[6] B. Rusten, A.K. Sahu, Microalgae growth for nutrient recovery from sludge liquor and

production of renewable bioenergy, Water Sci. Technol., 64 (2011) 1195-1201.

[7] N.F.Y. Tam, P.S. Lau, Y.S. Wong, Waste-water inorganic N-removal and P-removal by

immobilized Chlorella-vulgaris, Water Sci. Technol., 30 (1994) 369-374.

19

[8] K. Larsdotter, Wastewater treatment with microalgae - a literature review, Vatten, 62 (2006)

31-38.

[9] I. Woertz, A. Feffer, T. Lundquist, Y. Nelson, Algae grown on dairy and municipal wastewater

for simultaneous nutrient removal and lipid production for biofuel feedstock, J. Environ.

Eng.-ASCE, 135 (2009) 1115-1122.



[11] A. Widjaja, C.-C. Chien, Y.-H. Ju, Study of increasing lipid production from fresh water

microalgae Chlorella vulgaris, Journal of the Taiwan Institute of Chemical Engineers, 40

(2009) 13-20.

[12] R.B. Fischer, D.G. Peters, Basic theory and practice of quantitative chemical analysis.,

Third edition ed., W. B. Saunders Company., Philadelphia, London, Toronto, 1968.

[13] A.P.H.A., Standard methods for the examination of water and wastewater., Twenty first

Edition ed., American Water Works Association and Water Pollution Control Federation,

New York, 2005.

[14] Y. Azov, Effect of pH on inorganic carbon uptake in algal cultures, Applied and

Environmental Microbiology, 43 (1982) 1300-1306.

[15] H.B.A. Prins, J.F.H. Snel, R.J. Helder, P.E. Zanstra, Photosynthetic HCO- Utilization and

OH- Excretion in Aquatic Angiosperms., Plant Physiology 66 (1980) 818-822.

[16] Y. Shiraiwa, A. Goyal, N.E. Tolbert, Alkalization of the medium by unicellular green algae

during uptake dissolved inorganic carbon Plant and Cell Physiology, 34 (1993) 649-657.

[17] L. Christenson, R. Sims, Production and harvesting of microalgae for wastewater treatment,

biofuels, and bioproducts, Biotechnol. Adv., 29 (2011) 686-702.

20

[18] W. Zhou, Y. Li, M. Min, B. Hu, P. Chen, R. Ruan, Local bioprospecting for high-lipid

producing microalgal strains to be grown on concentrated municipal wastewater for

biofuel production, Bioresource Technol., 102 (2011) 6909-6919.

21

Growth of Chlorella Vulgaris using Sodium Bicarbonate under No Chapter 3

Mixing Condition2

3.1. Introduction

Microalgae have received recent attention due to their capability to produce a

considerable amount of lipids (e.g., 20-50% dry cell weight) for biodiesel production with fast

growth rates using a high photosynthetic efficiency and CO2 under autotrophic growth [1-7].

Microalgae are comprised of an elemental carbon composition of approximately 50 %(wt), but

little has been reported about the effects of inorganic carbon concentrations on their growth and

lipid formation [8, 9]. The growth of microalgae under different inlet CO2(g) concentrations was

previously studied in closed culture systems [10]. An optimum inlet CO2(g) concentration in the

range of 0.037 and 20%(v) was studied under a closed system. The growth of microalgae was

also investigated under different inlet CO2(g) concentrations in closed photobioreactors [11, 12].

These closed systems can increase the solubility of CO2(g) in the culture media by increasing a

partial pressure of CO2(g) in the gas phase. However, the pH of the culture media in closed

systems dramatically decreases with an increase in dissolved CO2(g) concentration, and a cost

for such closed systems is prohibitively expensive (i.e. at least ~4 times higher than open pond

systems) for large-scale cultivation [13, 14].

Meanwhile, when NaHCO3 is added to the culture media, it can readily generate a high

concentration of HCO3- with a small pH increase due to its high solubility (96 g/L H2O at 20 ºC).

In addition, a buffer capacity of sodium ion can help supersaturate dissolved inorganic carbon


content in this chapter has been published in Kim, J.; Lee, J.-Y.; Ahting, C.; Johnstone, R.; Lu, T., Growth of

Chlorella vulgaris using sodium bicarbonate under no mixing condition. Asia-Pacific Journal of Chemical

Engineering 2014, n/a-n/a.

22

concentrations and keep the dissolved CO2(g) concentration constant under an atmospheric

pressure for its use in open systems. Among the inorganic carbon species of CO2(aq), H2CO3,

HCO3-, and CO3

2-, microalgae can use only HCO3

- and CO2(aq) for their autotrophic growth with

a help of carbonic anhydrase and these two carbon species are denoted by DIC hereafter [9, 15].

Therefore, in this study, NaHCO3 was used as an inorganic carbon source for autotrophic

growth. A primary objective of this study is to investigate the autotrophic growth of Chlorella

vulgaris under different DIC concentrations controlled with sodium bicarbonate in the absence of

agitation of the culture media. Agitation was not used in this study to focus on the effects of

dissolved inorganic carbon concentrations on the growth by separating the potential effects of

light availability derived from mixing on suspended cells for photosynthesis. A DIC mass

balance in conjunction with external mass transfer of DIC (=HCO3- + CO2(aq)) was taken into

account in order to determine the DIC concentrations on the algal cell surface for the DIC mass

transfer from the aqueous phase (i.e. culture medium) to the solid phase (i.e. algal cell).

3.2. Material and Methods

3.2.1. Culture Medium and Conditions

C. vulgaris used (#2714) in this study was obtained from UTEX at the University of

Texas at Austin. A culture medium was prepared by following the modified Shuisheng-4

medium and 1,800 mL of the prepared medium was added in a 2-L bottle (21 cm (height) × 11

cm (diameter)) [16]. The cell density of Chlorella vulgaris in the wastewater medium was found

to be 67.30±0.48 mg/L after inoculation. The culture medium was mixed together with 30 mg/L

of calcium phosphate monobasic monohydrate (Ca(H2PO4)2·H2O), 200 mg/L of ammonium

sulfate ((NH4)2SO4), 80 mg/L of magnesium sulfate hetahydrate (MgSO4·7H2O), 25 mg/L of

23

potassium chloride (KCl), 1.5 mg/L of iron chloride(FeCl3), 10 mg/L of potassium phosphate

dibasic (K2HPO4), and 1 mL of A5 liquid. Herein, A5 liquid was prepared by mixing together

with 2.86 g/L of boric acid (H3BO3), 1.81 g/L of manganese chloride (MnCl2·4H2O), 0.222 g/L

of zinc sulfate (ZnSO4·7H2O), 0.391 g/L of sodium molybdate (Na2MO4·2H2O), 0.079 g/L of

cupric sulfate (CuSO4·5H2O).

The initial DIC concentrations in culture media were controlled by adding different

amounts of NaHCO3: 100 mg of NaHCO3/L H2O (C1 culture), 200 mg of NaHCO3/L H2O (C2

culture), 400 mg of NaHCO3/L H2O (C3 culture), 800 mg of NaHCO3/L H2O (C4 culture) and

2,400 mg of NaHCO3/L H2O (C5 culture). Then, a HCl solution was added to culture media to

control the initial pH of the media at 7.

During the culture, fluorescent lamps with 6,500-K color temperature similar to natural

sunlight were used as a source of light. An incoming light intensity to beakers was set to 6,000

lux (100.8 µmol∙m-2

∙s-1

), and the media were not mixed for open systems. The light intensity

was measured using a light intensity meter (HQRP digital lux meter, LX1010BS, Osprey-Talon

Company), and a 16-hr light and 8-hr dark cycle was applied to the culture. Then, the pH of the

culture media was controlled once daily by using an HCl solution.

3.2.2. Determination of Cell Mass Density of C. vulgaris

The cell mass density of C. vulgaris was determined by measuring the optical density at

682 nm for every 24 hours by using a UV-vis spectrophotometer (UV-1800, Shimadzu Scientific

Instruments) [17]. The absorbance of the UV spectrophotometer at 682 nm was calibrated by

measuring the weight of dried C. vulgaris. Then, the weight of dried biomass was obtained from

the prepared calibration curve.

24

A hemocytometer counting chamber was used to determine the cell number density of C.

vulgaris (i.e. the number of cells per unit volume of a sample) for the estimation of a total

external surface area of C. vulgaris in the culture mediums [18, 19]. For the estimation of a total

surface external surface, C. vulgaris was assumed to be completely spherical in shape. The

volume of each rectangular chamber in the hemocytometer was 0.1 mm3 (1 mm×1 mm×0.1 mm),

and the number of C. vulgaris cells in each chamber was counted under a microscope (Nikon

Labophot-2). Then an average cell number density was obtained by taking an average of the cell

number densities in all the rectangular chambers.

3.2.3. Determination of DIC Concentration

An acid-base titration method was used to determine the concentrations of inorganic

carbon species present in the aqueous phase [20]. This titration method determines a TIC

(=[HCO3-]+[CO3

2-]+[H2CO3]+[CO2(aq)]) at the pH end point of 4.3 in a 15-mL sample using 0.1

N and 0.01 N HCl solutions for the titration of high and low carbon concentrations, respectively.

Then, the DIC concentrations including CO2(aq) and HCO3- were determined by using the

equilibrium relations among HCO3-, CO3

2- and H2CO3 and CO2(aq). The accuracy of this

titration method was ensured by comparing a known amount of TIC dissolved from sodium

bicarbonate with the amount of DIC determined by the acid-base titration method. All TIC

concentrations were measured in duplicate.

25

3.2.4. Analytical Methods

The two coefficients of A and B in the Cs(t) function were determined by minimizing the

objective function f in Eq. (14) in comparison of the calculated DIC concentrations from Eq. (13)

with the measured DIC concentrations.


3.3.1. Use of Sodium Bicarbonate for Increasing DIC Concentrations

There are two methods that can be considered to supply a sufficient amount of DIC

concentrations to microalgae in the aqueous phase. One method is to increase a partial pressure

of CO2 gas in the gas phase using closed systems. The other one is to use a chemical with a CO2

buffer capacity that can keep DIC concentrations high for open systems. Equilibrium

calculations were made in order to demonstrate the partial pressures of CO2 gas and equilibrium

pH values that generate DIC concentrations comparable to those as a result of the dissolution of

NaHCO3. When CO2 gas absorbs into water, the following equilibrium relation and charge

balance Eqs. (1)-(5) apply:

(1)

(2)

(3)

(4)

(5)

'

2

2

2

** ' -2 2 3

2 2 2 3

CO

[H CO ]CO (g)+H O H CO , (=3.4×10 mol/(L×atm)=

P

COH

COH

1

+ -* + - -7 3

2 3 3 1 *

2 3

[H ][HCO ]H CO H +HCO , (=4.5×10 mol/L)=

[H CO ]CK

CK

2

+ 2-- + 2- -11 33 3 2 -

3

[H ][CO ]HCO H +CO , (=4.7×10 mol/L)=

[HCO ]CK

CK

+ - -14 2 2 + -

2H O H +OH , (=1×10 mol /L )=[H ][OH ]CWK

CWK

+ - 2- -

3 3[H ]=[HCO ]+2[CO ]+[OH ]

26

where all equilibrium constant values were obtained at 25 C [20]. The equilibrium

concentrations of , , , , and were determined and

summarized as shown in Table 3.1. The results show that a maximum DIC concentration under

1 atm comprising pure CO2 gas can have 396 mg carbon (C)/L with an equilibrium pH value of

3.9.

Table 3.1 DIC concentrations generated from CO2(g) absorption and NaHCO3 dissolution

at 1 atm.

pCO2 DIC (mg/L) Equilibrium

pH NaHCO3 (mg/L) DIC (mg/L)

Equilibrium

pH

0.037% 0.2 5.6 1.3 0.2 7.7

25% 101.9 4.2 713.2 101.0 8.3

50% 201.8 4.1 1,411.6 199.8 8.3

75% 300.0 4.0 2,097.9 296.9 8.3

100% 396.4 3.9 2,772.7 392.4 8.3

On the other hand, when NaHCO3 dissolves in water, the HCO3- in NaHCO3 is

distributed among H2CO3*, HCO3

-, and CO3

2- depending on an equilibrium pH value. The

NaHCO3 solution system constructs the following equilibrium relations in Eqs. (2)-(4), mass

balance, and charge balance Eqs. in (6)-(8) used to determine the DIC concentrations and

equilibrium pH values:

(6)

(7)

(8)

*

2 3 2 3 2H CO (=H CO +CO (aq))+H

-OH -

3HCO 2-

3CO

+ 3

3

g of NaHCO /L[Na ]=

M.W. of NaHCO (=84.007g/mol)

+ - 2- *

3 3 2 3[TIC]=[Na ]=[HCO ]+[CO ]+[H CO ]

+ + - 2- -

3 3[H ]+[Na ]=[HCO ]+2[CO ]+[OH ]

27

The concentrations of Na+, H2CO3

*, H

+, OH

-, HCO3

-, and CO3

2- can be determined from the

above six equations as shown in Table 3.1. It shows that the dissolution of ~2.8 g/L of NaHCO3

can generate ~400 mg/L of DIC, which is comparable to a maximum DIC concentration that can

be generated with pure CO2 gas at 1 atm. The equilibrium pH value is also kept at a constant

value of 8.3 within a benign pH window where C. vulgaris is reported to grow well [21, 22].

These equilibrium calculations indicate that NaHCO3 may be able to be used for open pond

systems as a buffer solution that can increase the supply of DIC.

3.3.2. Growth of C. vulgaris in NaHCO3 Solutions

Figure 3.1 Growth of C. vulgaris in different initial DIC concentrations.

Based on the above equilibrium calculations made for NaHCO3 solutions, C. vulgaris

was cultivated in five different initial DIC concentrations between 17 and 361 mg C/L. The

highest DIC concentration of 361 mg C/L was almost comparable to a maximum DIC

concentration that can be dissolved with pure CO2 gas at 1 atm. Figure 3.1 shows the growth

rates of C. vulgaris under different initial DIC concentrations (17, 33, 62, 123, and 361 mg C/L),

28

and Figure 3.2 shows the DIC concentrations in the culture media. During the autotrophic

growth of C. vulgaris, the pH values of the culture media continued to increase as a result of the

uptake of HCO3- ion and thus the generation of OH

- ion. The increased pH value was controlled

at 7 by adding an HCl solution once daily, where C. vulgaris was observed to grow well within a

pH range of 7 to 8 in our lab and was also previously reported [22]. Instead of CO2 gas, an HCl

solution was used to track the temporal changes in DIC concentrations in the culture media. The

growth of C. vulgaris for C1, C2, and C3 cultures stopped almost after 48, 72, and 120 hrs,

respectively, after all DIC concentrations were consumed. On the other hand, the DIC

concentrations for C4 and C5 cultures were available until 144 hrs, and their growth continued.

It is interesting to note that the growth rates for C4 and C5 cultures were comparable although a

difference in DIC concentrations in the media is more than twice over the entire culture period.

This result suggests that the external DIC mass transfer by molecular diffusion between the bulk

aqueous and solid phases is unlikely to be a rate-limiting step for the growth in the absence of

agitation.

29

Figure 3.2 Temporal DIC concentration profiles in culture media with different initial DIC

concentrations.

3.3.3. External Mass Transfer of DIC from Bulk culture Medium to Cell Surface

Based on the above growth results, the DIC concentrations on the cell surface were

estimated to determine the mass transfer of DIC from the bulk aqueous phase to the solid phase

(i.e. from culture medium to cell surface) under different initial DIC concentrations. For the

estimation, C. vulgaris was assumed to be spherical in shape with a uniform 3-µm diameter

based on our observation under the microscope and an average value reported in the literature

[23, 24].

A DIC mole balance equation was taken to describe the DIC uptake by C. vulgaris in a

batch reactor as shown in Eq. (9).

(9)

where Vl is the liquid volume (m3), CB(t) is the bulk DIC concentration (mol/m

3) in the culture

medium, kc is the mass transfer coefficient (m/s), A(t) is the total surface area of C. vulgaris cells,

and CS(t) is the surface concentration of DIC (mol/m3). 0.00018 m

3 was used for Vl and DIC

concentrations in the culture media were measured once daily for CB(t). The total cell surface

area, A(t), increased with an increase in cell growth over time, and was determined by using the

cell density data shown in Eq. (10).

(10)

The total cell surface was calculated from a cell mass density (g/m3) of C. vulgaris determined

from UV-vis spectroscopy, a cell count (4.6×1010

/g) determined by a hemocytometer counting

( )( )( ( ) ( ))B

l c B S

dC tV k A t C t C t

dt

2cell number surface area (m )( )= × ×medium volume (ml)

sample volume (ml) cellA t

30

chamber, a single cell external surface area of 2.83×10-11

m2 for an algal cell with a 3-µm

diameter, and a culture medium volume of 1.8 L.

Table 3.2 Coefficients of a third-order polynomial function in Eq. (11) for total surface

area.

Coefficient C1 medium C2 medium C3 medium C4 medium C5 medium

A 1.6 × 10-1

1.5 × 10-1

1.5 × 10-1

1.5 × 10-1

1.5 × 10-1

B 1.2 × 10-3

2.4 × 10-3

1.7 × 10-3

6.9 × 10-4

8.8 × 10-4

C -9.4 × 10-8

-9.8 × 10-6

1.7 × 10-6

3.7 × 10-5

3.1 × 10-5

D 2.0 × 10-8

3.5 × 10-9

-1.0 × 10-7

-1.3 × 10-7

-9.1 × 10-8

Then, a third-order polynomial function in Eq. (11) was constructed to express the total surface

area, A(t). The coefficients of A, B, C and D for the total surface area for all C1C5 cultures are

summarized in Table 3.2.

(11)

The liquid-solid mass-transfer coefficient (kC) was determined using the Brian and Hales

equation in Eq. (12) [25].

(12)

where DDIC,water is a diffusivity value for DIC at 25 C; dp indicates a 3-µm diameter of an algal

cell; Rep is the Reynolds number; Pe is the Peclet number; Sc is the Schmidt number and Sh

indicates Sherwood number. A diffusivity value of 1.1×10-9

m2/s was used for the diffusion of

DIC in water [26, 27]. In the absence of agitation, the Sherwood number becomes a limiting

value of 2 valid for diffusion, from which 7.3×10-4

(m/s) was determined as a mass-transfer

coefficient.

2 3( )A t A Bt Ct Dt

2/3 1/2,

,

Sh= =(4+1.21Pe ) , (Pe=Re ScPe 10,000)c p

p

DIC water

k d

D

31

A linear function was used to determine the surface DIC concentration, Cs(t), as shown in Eq.

(13).

(13)

The coefficients of A and B are summarized in Table 3.3.

(14)

Table 3.3. Coefficients used for surface DIC concentrations in Eq. (13).

Coefficient C1 medium C2 medium C3 medium C4 medium C5 medium

A -3.0 × 10-6

-9.1 × 10-6

-3.6 × 10-5

-5.2 × 10-5

-8.8 × 10-5

B 7.6 × 10-4

1.8 × 10-3

4.8 × 10-3

1.1 × 10-2

3.0 × 10-2

( )sC t At B

2

exp ( ) ( )cal

B B

t

f C t C t

32

Figure 3.3 (a) Temporal DIC concentration profile at the cell surface in culture media with

different initial DIC concentrations (b) Comparison of calculated bulk DIC concentrations

with measured bulk DIC concentrations, and (c) Diffusive flux of DIC.

The calculated surface DIC concentrations are shown in Figure 3.3(a). The surface DIC

concentrations (CS(t)) are slightly less than the bulk DIC concentrations (CB(t)) due to small DIC

mass transfer rates under no mixing condition. The calculated bulk DIC concentrations with the

estimated surface DIC concentrations are in good agreement with the measured bulk DIC

concentrations as shown in Figure 3.3(b). The calculated diffusive flux of DIC shows a decrease

over time for all cultures. Cultures in high DIC concentrations showed slightly high DIC fluxes,

but overall the effect was not significant under no mixing condition as shown in Figure 3.3(c).

This result reconfirms that the external DIC mass-transfer resistance by diffusion between the

bulk aqueous and solid phases is not significant, and suggests that the DIC consumption rate

inside the algal cell is very likely to be a rate-limiting step for the growth in the absence of

agitation.

3.4. Conclusions

In this study, the growth of C. vulgaris in terms of different initial DIC concentrations

under no mixing condition was investigated using NaHCO3 as an inorganic carbon source.

NaHCO3 was found to be used as a buffer that can help keep the DIC concentrations in the

culture medium high and the pH of the medium suitable for algal culture. When a DIC

concentration using NaHCO3 was generated at a level similar to that under pure CO2 gas,

NaHCO3 did not show any adverse impact on the growth. It was found that the growth of C.

33

vulgaris under no mixing condition is not significantly influenced by different DIC concentration

levels available in the culture media. The estimated DIC concentrations at the algal cell surface

were also comparable to the bulk DIC concentrations. These results indicate that the external

mass transfer of DIC from the culture medium to the cell is not a rate-limiting step for the growth

and intracellular mass transfer and/or photosynthesis inside the algal cell is very likely to be a

rate-limiting step under no mixing condition. The effects of DIC concentrations on the growth

under mixing conditions plan to be investigated.


This study was supported by the Metropolitan Sewer District of Greater Cincinnati

(MSDGC) under Master Services Agreement No. 85X10431, Task Order No. 0210000209.

During this study, Christina Ahting and Rachel Johnston were supported by NSF Grant No.

EEC-1004623 for REU Site on Sustainable Urban Environments. The authors appreciate their

financial support.

3.6. References

[1] T. Burton, H. Lyons, Y. Lerat, M. Stanley, M.B. Rasmussen, A review of the potential of

marine algae as a source of biofuel in Ireland, in, Dublin: Sustainable Energy Ireland-SEI,

2009.

[2] R. Davis, A. Aden, P.T. Pienkos, Techno-economic analysis of autotrophic microalgae for

fuel production, Applied Energy, 88 (2011) 3524-3531.

34




[4] Y. Li, D. Han, M. Sommerfeld, Q. Hu, Photosynthetic carbon partitioning and lipid

production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under

nitrogen-limited conditions, Bioresource Technology, 102 (2011) 123-129.

[5] Y.Q. Li, M. Horsman, B. Wang, N. Wu, C.Q. Lan, Effects of nitrogen sources on cell growth

and lipid accumulation of green alga Neochloris oleoabundans, Applied Microbiology

and Biotechnology, 81 (2008) 629-636.

[6] X. Miao, Q. Wu, Biodiesel production from heterotrophic microalgal oil, Bioresource

Technology, 97 (2006) 841-846.

[7] Á. Sánchez, R. Maceiras, Á. Cancela, A. Pérez, Culture aspects of Isochrysis galbana for

biodiesel production, Applied Energy, 101 (2013) 192-197.

[8] L. Jiang, S. Luo, X. Fan, Z. Yang, R. Guo, Biomass and lipid production of marine

microalgae using municipal wastewater and high concentration of CO2, Applied Energy,

88 (2011) 3336-3341.

[9] L. Lardon, A. Helias, B. Sialve, J.P. Stayer, O. Bernard, Life-cycle assessment of biodiesel

production from microalgae, Environmental Science and Technology, 43 (2009) 6475-

6481.



35

[11] L. He, V.R. Subramanian, Y.J. Tang, Experimental analysis and model-based optimization

of microalgae growth in photo-bioreactors using flue gas, Biomass and Bioenergy, 41

(2012) 131-138.

[12] D. Sasi, P. Mitra, A. Vigueras, G.A. Hill, Growth kinetics and lipid production using

Chlorella vulgaris in a circulating loop photobioreactor, J. Chem. Technol. Biotechnol.,

86 (2011) 875-880.

[13] L. Amer, B. Adhikari, J. Pellegrino, Technoeconomic analysis of five microalgae-to-

biofuels processes of varying complexity, Bioresource Technology, 102 (2011) 9350-

9359.

[14] M. Odlare, E. Nehrenheim, V. Ribé, E. Thorin, M. Gavare, M. Grube, Cultivation of algae

with indigenous species – Potentials for regional biofuel production, Applied Energy, 88

(2011) 3280-3285.

[15] M. Giordano, J. Beardall, J.A. Raven, CO2 concentrating mechanisms in algae: Mechanisms,

environmental modulation, and evolution., Annual Review of Plant Biology, 56 (2005)

99-131.

[16] Y.X. Zhou, Z.S. Zhang, The toxicity methods in aquatic living., Agriculture Press, Beijing,

1989.



(2009) 13-20.

[18] J.W. Rachlin, M. Farran, Growth response of the green algae Chlorella vulgaris to selective

concentrations of zinc, Water Research, 8 (1974) 575-577.

36

[19] J.W. Rachlin, A. Grosso, The effects of pH on the growth of Chlorella vulgaris and its

interactions with cadmium toxicity, Archives of environmental contamination and

toxicology, 20 (1991) 505-508.








[23] S. Hadjoudja, V. Deluchat, M. Baudu, Cell surface characterisation of Microcystis

aeruginosa and Chlorella vulgaris, Journal of colloid and interface science, 342 (2010)

293-299.

[24] B.G. le Grooth, T.H. Geerken, J. Greve, The cytodisk: A cytometer based upon a new

principle of cell alignment, Cytometry, 6 (1985) 226-233.

[25] P.L.T. Brian, H.B. Hales, Effects of transpiration and changing diameter on heat and mass

transfer to spheres, AIChE Journal, 15 (1969) 419-425.

[26] S. Chatterjee, G.R. Dickens, G. Bhatnagar, W.G. Chapman, B. Dugan, G.T. Snyder, G.J.

Hirasaki, Pore water sulfate, alkalinity, and carbon isotope profiles in shallow sediment

above marine gas hydrate systems: A numerical modeling perspective, Journal of

Geophysical Research, 116 (2011) B09103.

[27] L. Yuan-Hui, S. Gregory, Diffusion of ions in sea water and in deep-sea sediments,

Geochimica et cosmochimica acta, 38 (1974) 703-714.

37

Effects of Dissolved Inorganic Carbon and Mixing on Autotrophic Chapter 4

Growth of Chlorella vulgaris3

4.1. Introduction

Microalgae recently drew significant attention due to a growing demand for petroleum

[1]. They are reported to have a substantial amount of lipids (approximately 15-50% dry cell

weight), faster growth rates than terrestrial plants owing to their high photosynthesis rates, and a

large fraction of solar energy utilization (3-10%) [2-4]. Microalgae can utilize CO2 gas, which is

a major contributor to global warming, as an inorganic carbon source for their autotrophic

growth [5, 6].

Open and closed systems can be used to cultivate microalgae using CO2(g) [7]. An open

system is simple and easy to scale up at a relatively low cost for large-scale cultivation.

However, the production of DIC (= HCO3- + CO2(aq)) species derived from CO2(g) dissolution

is limited in the open pond system due to a low solubility of CO2(g) under an atmospheric

pressure and the DIC concentration available in water may not be high enough to meet a desired

microalgal cultivation rate. Meanwhile, a closed system can readily increase the DIC

concentration at a desired level by increasing the pCO2. However, the cost of such a closed

system is expensive for a large-scale cultivation system [8]. In addition, the pH of the culture

medium decreases with an increase in DIC concentrations as a result of CO2(g) dissolution,

which interferes with an allowable pH window for microalgal growth [9, 10]. In this respect, it


content in this chapter has been published in Kim, J.; Lee, J.-Y.; Lu, T., Effects of dissolved inorganic carbon and

mixing on autotrophic growth of Chlorella vulgaris. Biochemical Engineering Journal 2014, 82 (0), 34-40.

38

is important to study whether microalgal growth can be limited by DIC concentrations for large-

scale cultivation.

In this study, NaHCO3 was used as a buffer that could keep the DIC concentration level

high in the culture medium. The primary objectives of this study were to examine the feasibility

of using NaHCO3 as a buffer to overcome potential DIC limitation for open systems and to keep

the pH of the medium within a desired pH window particularly for closed systems during the

autotrophic growth of C. vulgaris. The effects of DIC concentrations with NaHCO3 on the

growth were investigated by varying different mixing speeds for the consideration of mass

transfer and photosynthetic reaction.


4.2.1. Culture Media and Conditions

C. vulgaris (Beij. [K&H]) used (UTEX #2714) in this study was obtained from UTEX at

the University of Texas at Austin. A culture medium was prepared by following Shuisheng-4

medium and 1,800 mL of the prepared medium was added to a 2-L bottle (21 cm (height) × 11

cm (diameter)) [11]. The cell density of C. vulgaris in the medium was found to be 117.1±0.6

(C1, C2, C3, C4, C5 and C6) and 46.6±0.2 (D1, D2, D3, D4, and D5) mg/L after inoculation.

NaHCO3 was not added to the medium during this step. Then, an initial DIC concentration in a

culture medium was controlled by adding a different amount of NaHCO3: 100 mg of NaHCO3/L

for C1, C2, and C3 cultures; 1,000 mg of NaHCO3/L for C4, C5, C6, D1 and D3 cultures; 5,000

mg of NaHCO3/L for D2 and D4 cultures; and 10,000 mg of NaHCO3/L H2O for D5 culture.

Then, CO2(g) was added to all the culture media to control the initial pH of the media at 7 and

this pH control was repeated once daily. During the culture, C1, C4, D1 and D2 culture media

39

were not mixed. C2, C5, D3, D4 and D5 culture media were mixed by using a magnetic stirrer at

a speed of 125 rpm. C3 and C6 media were mixed at a speed of 550 rpm.

During the culture, fluorescent lamps with 6,500-K color temperature similar to natural

sunlight color temperature were used as a light source. The incident light intensity at the reactor

surface was set to 6,000 lux (100.8 µmol/(m2∙s)), and a 16-hr light and 8-hr dark cycle was

applied to all the cultures. All the culture media were open to an atmosphere at 25 ºC.

4.2.2. Determination of Cell mass and Number Densities of C. vulgaris

The cell mass density of C. vulgaris in the culture medium was determined by measuring

the optical density at 682 nm by using a UV-vis spectrophotometer (UV-1800, Shimadzu

Scientific Instruments). The absorbance of the UV-vis spectrophotometer at 682 nm was

calibrated by measuring the weight of dried C. vulgaris [12]. Then, the weight of the dried

biomass was obtained from the prepared calibration curve.

A hemocytometer counting chamber was used to determine the cell number density of C.

vulgaris (i.e. the number of cells per unit volume of a sample) for the estimation of a total

external surface area of C. vulgaris in the culture mediums [13, 14]. The volume of each

rectangular chamber in the hemocytometer was 0.1 mm3 (1 mm×1 mm×0.1 mm), and the

number of C. vulgaris cells in each chamber was counted under a microscope (Nikon Labophot-

2). Then an average cell number density was obtained by taking an average of the cell number

densities in all the rectangular chambers.

40

4.2.3. Determination of DIC, NH3/NH4+, and PO4

3- Concentrations

An acid-base titration method was used to determine the concentrations of DIC species

present in the aqueous phase [15]. This titration method determines a total inorganic carbon

(TIC) concentration (=[HCO3-]+[CO3

2-]+[H2CO3]+[CO2(aq)]) in a 15-mL sample using 0.1 N

and 0.01 N HCl solutions for the titration of high and low carbon concentrations, respectively.

Then, the DIC concentrations were calculated by using the equilibrium relations shown in the

next section among HCO3-, CO3

2-, H2CO3, and CO2(aq).

The concentrations of NH3/NH4+ were determined using an ammonia probe (Model:

9512HPBNWP Orion Thermo Scientific) and PO43-

was determined using Phosver 3 phosphate

regent (Hach company) [16].

4.2.4. Determination of Carbon Content in C. vulgaris

The carbon content in C. vulgaris was analyzed by an elemental analyzer (Vario Macro

cube, Elementar Americas, Inc.). The content was used to estimate the specific DIC uptake rate

by C. vulgaris with respect to different DIC concentrations in the culture media.


4.3.1 Comparison of Equilibrium DIC Concentrations with and without NaHCO3

The DIC concentration in the aqueous phase is dependent on the pCO2 based on Henry’s

law. Then the dissolved *

2 3H CO will further dissociate into HCO3- bicarbonate and CO3

2- ions,

and the equilibrium concentrations for all species can be calculated by the following equilibrium

and charge balance eqns (1)-(5):

41

2

2

2

** ' 2 2 3

2 2 2 3

'[ ]

( ) , ( 3.4 10 / ( ))CO

CO

CO

H H COCO g H O H CO H mol L atm

P

(1)

* 7 312 3 3 1 *

2 3

[ ][ ], ( 4.5 10 / )

[ ]

H HCOKH CO H HCO K mol L

H CO

(2)

2

2 11 323 3 2

3

[ ][ ], ( 4.7 10 / )

[ ]

H COKHCO H CO K mol L

HCO

(3)

14 2 2

2 , ( 1.0 10 / ) [ ][ ]WW

KH O H OH K mol L H OH (4)

2 2

3 3[ ] [ ] 2[ ] [ ]H HCO CO OH (5)

where all the equilibrium constant values were obtained at 25 ºC. Here, *

2 3H CO consists of

2 3H CO and 2( )CO aq as shown in eqn (6), and these two components have the following

equilibrium relationship in eqn (7):

*

2 3 2 2 3[ ] ( )H CO CO aq H CO (6)

3 2 332 2 2 3 3

2

[ ]( ) , ( 2.6 10 / )

[ ( )]

H COKCO aq H O H CO K mol L

CO aq

(7)

where K3 was obtained at 25 ºC. Then, the equilibrium concentrations *

2 3H CO , 2 3H CO ,

2( )CO aq , H , OH

, 3HCO and

2

3CO can be determined, and the equilibrium pH values and

DIC concentrations with respect to pCO2 are summarized in Table 4.1. When CO2(g) is used to

increase the DIC concentration, pH decreases with an increase in the partial pressure of CO2(g)

(pCO2). A maximum DIC concentration under 1 atm pure CO2 gas is estimated to be 396 mg

carbon (C)/L with an equilibrium pH value of 3.9.

42

Table 4.1 DIC concentrations generated from CO2(g) absorption and NaHCO3 dissolution

at 1 atm.

pCO2 (%) DIC (mg/L) pH NaHCO3(mg/L) DIC (mg/L) pH

0.037 0.2 5.6 1.3 0.2 7.7

25 101.9 4.2 713.2 101.0 8.3

50 201.8 4.1 1,411.6 199.8 8.3

75 300.0 4.0 2,097.9 296.9 8.3

100 396.4 3.9 2,772.7 392.4 8.3

When NaHCO3 is used as a buffer to keep the DIC concentration high for open pond

systems, NaHCO3 readily dissociates into Na+ and HCO3

- ions. Then, HCO3

- is distributed to

2 3H CO , 2( )CO aq , and 2

3CO at equilibrium pH. Among these inorganic carbon species, the

DIC concentrations can be calculated by the above equilibrium relations in eqns (2)-(4), mass

balance for DIC species, and charge balance equations in eqns (8)-(10).

3

3

g of NaHCO /L[ ]

MW of NaHCO (=84.007g/gmol)Na (8)

2 *

3 3 2 3[ ] [ ] [ ] [ ] [ ]TIC Na HCO CO H CO (9)

2

3 3[ ] [ ] [ ] 2[ ] [ ]H Na HCO CO OH (10)

The equilibrium concentrations of Na, 2 3H CO , 2( )CO aq , H

, OH , 3HCO

, and 2

3CO can

be determined from the above relations. As shown in Table 4.1, the dissolution of ~2.8 g/L of

NaHCO3 can produce ~400 mg/L of DIC, which is comparable to a maximum DIC concentration

producible with pure CO2(g) at 1 atm. The equilibrium pH values are also kept in a range of 7-

8.3, which is within a pH range (7-9) suitable for the cultivation of microalgae such as Chlorella

sp., Dunaliella salina, and Duneliella sp. as reported in the literature [9, 10]. These results

indicate that NaHCO3 can be effectively utilized for open systems as a buffer that can increase

the production of DIC along with CO2(g).

43

4.3.2. Growth of C. vulgaris under Different DIC Concentrations and Mixing Conditions

Figure 4.1 Effects of different DIC concentrations and mixing conditions on the growth of

C. vulgaris: (a) initial DIC of 15 mg C/L under different mixing speeds; (b) initial DIC of

144 mg C/L under different mixing speeds; (c) pH change under different DIC and mixing

44

speeds. Note: CO2(g) was added to all culture media to control the pH of the media at 7

once daily; (d) nitrogen concentration at 192 hours under different DIC concentrations and

mixing speeds; (e) phosphorus concentration at 192 hours under different DIC

concentrations and mixing speeds.

C. vulgaris was cultivated in two different initial DIC concentrations of 15 and 144 mg

C/L using NaHCO3 under three different mixing speeds of 0, 125, and 550 rpm. During the

culture, the pH values of the culture media increased up to pH=8.6 as a result of additional OH-

ion generated by C. vulgaris (Figure 4.1(c)). The increased pH values were controlled at 7 by

adding CO2(g) once daily because C. vulgaris could grow best at pH 78 in our lab and was also

previously reported [9, 10]. During the culture, it was ensured that essential nutrients except for

DIC were sufficiently supplied by monitoring nitrogen and phosphorus concentrations as shown

in Figure 4.1(d) and (e). The same amount of extra nutrients in Shuisheng-4 medium including

nitrogen and phosphorus was added all media every 3 days except for inorganic carbon (i.e. DIC)

to ensure a sufficient supply of essential nutrients.

The growth for C1-C3 cultures under 15 mg C/L of a low initial DIC concentration

shown in Figure 4.1(a) exhibited gradual increases regardless of mixing speeds, but was found to

be insignificant. The two different mixing speeds between 125 and 550 rpm did not generate any

noticeable difference. A small difference in the growth under mixing (C2 and C3) and no

mixing (C1) conditions seems to be attributed to the photosynthetic kinetics under the low DIC

concentration. However, the growth for C4-C6 cultures with 144 mg C/L of a high initial DIC

concentration showed significant differences in terms of the presence of mixing as shown in

Figure 4.1(b). When the culture was not mixed (C1 and C4) or a DIC concentration was low (C2

45

and C3), the growths for C1-C4 did not show any significant difference. However, the growths

under the high DIC concentration with agitation (C5 and C6) were significantly higher than that

under the low DIC concentration with agitation (C2 and C3).

This significant difference could result from the differences in (1) external DIC mass-

transfer resistance between the medium and the cell surface, (2) intracellular DIC mass-transfer

resistance inside the cell, and (3) photosynthesis reaction kinetics. The external mass-transfer

resistances in terms of different mixing speeds can be analysed. However, it is very difficult to

separately analyse the intracellular mass-transfer resistance and the intrinsic photosynthetic

reaction kinetics due to the complexity and incomplete understanding of internal DIC mass

transfer through the complex internal cell structure, and a lack of kinetic expressions for the

breakdown of DIC through carbonic anhydrase followed by the photosynthetic reaction [17, 18].

Therefore, as a first step, the external mass-transfer resistances under different mixing speeds

were evaluated in the next section.

4.3.3. External Mass Transfer of DIC from Bulk Medium to Cell Surface

Table 4.2 Coefficients used for total surface area, A(t), and DIC surface concentrations,

CS(t).

Total cell surface area, 2 3( )A t A Bt Ct Dt

Coefficients C1 C2 C3 C4 C5 C6

A 2.7×10-1

2.5×10-1

2.5×10-1

2.6×10-1

2.7×10-1

2.7×10-1

B 3.1×10-3

3.3×10-3

2.3×10-3

5.0×10-3

-1.6×10-3

-2.8×10-3

C 7.4×10-6

2.8×10-5

3.8×10-5

-2.2×10-5

1.5×10-4

1.7×10-4

D -5.7×10-8

-1.5×10-7

-1.8×10-7

4.6×10-8

-3.1×10-7

-3.6×10-7

DIC surface concentration, ( )sC t At B

46

A -3.4×10-6

-4.2×10-6

-4.0×10-6

-6.5×10-6

-2.2×10-5

-1.9×10-5

B 1.2×10-3

1.3×10-3

1.3×10-3

1.2×10-2

1.2×10-2

1.2×10-2

The DIC concentration on the cell surface was estimated to determine the mass transfer

of DIC from the bulk medium to the cell surface under different DIC concentrations and mixing

speeds. For the estimation, our particle size measurement shows that C. vulgaris has a uniform

3-μm diameter. A DIC mole balance in eqn (11) was taken to describe the DIC uptake by C.

vulgaris in a batch reactor:

( )

( )( ( ) ( ))Bl c B S

dC tV k A t C t C t

dt (11)

where Vl is the liquid volume (m3), CB is the bulk concentration of DIC (mol/m

3); kc is the mass-

transfer coefficient (m/s); A(t) is the total surface area of C. vulgaris cells (m2); and CS(t) is the

surface concentration of DIC (mol/m3). 0.0018 m

3 was used for Vl and the measured DIC

concentrations in the culture media were used for CB(t). The total cell surface area, A(t),

increased with respect to time as a result of cell growth, and was determined by using the cell

number density data determined from hemocytometer measurements.

2cell number surface area (m )

A(t)= × ×medium volume (ml)sample volume (ml) cell

(12)

where a single cell external surface area of 2.83×10-11

m2 was used for an algal cell with an

average 3-μm diameter (i.e. 2D ), and 1,800 mL was used as a culture medium volume. Then,

a third-order polynomial function was found to correlate measured A(t) well, and the parameter

values are summarized in Table 4.2. The liquid-solid mass-transfer coefficient (kc) was

determined by using the equation of Brian and Hales in eqn (13) [19].

47

2/3 1/2,

,

(4 1.21 ) , ( Re 10,000)c p

p

DIC water

k dSh Pe Pe Sc Pe

D (13)

where DDIC,water is a diffusivity value of DIC in water at 25 ºC (1.1×10-9

m2/s); dp is an average

diameter of C. vulgaris cell (3 μm); Pe is the Peclet number, Rep is the particle Reynolds number;

Sc is the Schmidt number; and Sh is the Sherwood number [20-23]. The Schmidt number was

estimated to be 810.91 with a kinematic viscosity of water at 25 ºC (0.892×10-6

m2/s). The

particle Reynolds number was determined using eqn (14) for the movement of the algal cell in

the culture medium.

( )

Rep

p

u r d

(14)

where ( )u r is the average velocity of the algal cell moving in the direction with respect to the

radial direction induced by the rotational motion of a magnetic stirrer as shown in Figure 4.2.

For the estimation of the average velocity, the flow regime should first be determined by the

impeller Reynolds number in eqn (15) [24].

2

Rea

N D

(15)

where N is the impeller speed in rpm and D is the length of a magnetic stirrer in meter and is

the kinematic viscosity of water in m2/s. The flow is turbulent when the impeller Reynolds

number is greater than 10,000 [24]. The impeller Reynolds number was 6,075 and 26,729, and

thus the flow regimes were found to be laminar and turbulent under 125 and 550 rpm,

respectively.

48

Figure 4.2 A schematic of rotational motion generated by a magnetic stirrer in a batch

reactor.

The rotational motion of a magnetic stirrer creates the forced vortex and free vortex

regions in an unbaffled stirred tank as shown in Figure 4.2. In the forced vortex region, the

tangential velocity ( u ) linearly increases with the agitator velocity independent of the flow

regimes of laminar and turbulent flows [24]. In the free vortex region, u is different under

laminar and turbulent flow regimes and is determined by the following relations [25-27].

0 , ( ) (Forced vortex for laminar & turbulent)c ar r u r rw (16)

2

2 2

( )( ), ( ) (Free vortex for laminar)

( 4 )

ac

D r R r R wr r R u r

r D R

(17)

49

2

, u = (Free vortex for turbulent)c ac

r wr R

r (18)

Then the average velocity can be determined by eqn (19).

0

2

( )2( ) (Average velocity)

Ru r rdr

u rR

(19)

where cr is the radius of the forced vortex; wa is the agitator velocity (e.g. 125 rpm =13.09/s); D

is the length of a magnetic bar (0.051 m); R is the reactor radius (0.12m); and u(r) is the local

velocity profile in the direction with respect to the radial direction. The velocity profile of u(r)

in eqns (16) and (18) were suggested by Nagata et al., and the one in eqn (17) was derived from

the Navier-Stokes equation [24-27]. The film thickness between the medium and cell surface

was estimated to range between 0.21 and 1.5 m using eqn (20). The determined average

velocity ( ( )u r ), Reynolds number (Re), Sherwood number (Sh), Peclet number (Pe), and the

film thickness (δ) are summarized in Table 4.3.

,DIC water

c

D

k (20)

50

Table 4.3 Impeller Reynolds, flow regime, average velocity, Reynolds number, Schmidt

number, Peclet number, Sherwood number, mass transfer coefficient, and film thickness in

terms of mixing speed.

Culture Mixing (rpm) Rea Flow u (m/s)

C1 & C4 0 0 No flow 0

C2 & C5 125 6,075 Laminar 0.078

C3 & C6 550 26,729 Turbulent 0.536

Culture Rep Sc Pe Sh kc (m/s) δ (m)

C1 & C4 0 810.91 0 2 7.3×10-4

1.5×10-6

C2 & C5 0.26 810.91 212.73 6.86 2.5×10-3

4.4×10-7

C3 & C6 1.8 810.91 1,461.19 12.64 5.3×10-3

2.1×10-7

The temporal DIC concentrations on the cell surface, CS(t), in eqn (11) were calculated

by constructing a linear function. The two parameter values of A and B in the CS(t) function

shown in Table 4.2 were determined by minimizing the objective function, f, in eqn (21) by

comparing the calculated DIC concentrations with the measured bulk DIC concentrations.

2

exp ( ) ( )cal

B Bt

f C t C t (21)

51

Figure 4.3 Comparison of bulk DIC concentrations (CB) with surface DIC concentration

(CS) in terms of different initial DIC concentrations and mixing speeds at 192 hours.

The calculated surface DIC concentrations, CS(t), are co-plotted with the measured DIC

concentrations at 192 hours in Figure 4.3. For all of C1-C6 cultures, the bulk medium and

surface DIC concentrations were very close. For C1-C3 cultures with a low initial DIC

concentration of 15 mg C/L, the bulk and surface DIC concentrations did not show any

significant differences regardless of a mixing speed. However, for C4-C6 cultures with a high

initial DIC concentration of 144 mg C/L, significant amounts of DIC concentrations were found

to be consumed under mixing conditions, resulting in a significant difference in growth as shown

in Figure 4.1. This result validates that the external mass-transfer resistance is not a rate-limiting

step for the growth, and the growth is primarily limited by the photosynthesis reaction and/or

intracellular DIC mass transfer. Both high DIC concentration and mixing were found to be

52

required for enhanced cell growth because photosynthesis comes into effect under cell

suspension requiring agitation and then start to actively consume DIC.

4.3.4. Effects of DIC Concentration at Cell Surface on DIC Uptake Rate of C. vulgaris

Figure 4.4 Specific DIC uptake rate of C. vulgaris with respect to different DIC

concentrations (15, 30, 144, and 712 mg C/L) at the cell surface.

Little has been reported about mass transport within the cell (i.e. intracellular transport)

and carbon fixation for the photosynthesis reaction. Thus, there is a lack of mechanistic

understanding and kinetic expressions for intracellular DIC transport and subsequent carbon

fixation particularly for the autotrophic growth of microalgae [18]. As a second step, the specific

DIC uptake rate was used as a simple means to evaluate the effects of different DIC

concentrations at the cell surface on the observed photosynthesis kinetics for intracellular mass

transport combined with photosynthesis. As shown in Figure 4.4, the specific DIC uptake rate

53

with respect to different DIC concentrations (0, 15, 30, 144, and 712 mg C/L) in the culture

media under mixing (125 rpm) was estimated by multiplying the cell density increase of C.

vulgaris during the growth phase (24-192 h for initial DIC concentrations of 15, 30, and 144 mg

C/L and 72-192 h for initial DIC concentrations of 712 mg C/L) by the carbon content of the

cells determined for all cultures after 192 h. The carbon content in dried C. vulgaris was

determined to be 50±2%(wt) for all cultures by elemental analysis, and thus 50%(wt) was

selected as carbon content for all cultures. The specific DIC uptake rate was found to reach a

maximum at 144 mg C/L, and the rate did not significantly increase any further with a DIC

concentration under a given light intensity condition. Many previous studies reported that

microalgal growth at high cell densities is ultimately limited by light availability [28-30]. These

results suggest that the growth is initially limited by DIC concentration at low cell densities and

then most likely starts to be limited by light availability due to high cell densities called “shading

effect or photolimitation”. This also indicates that the use of sodium bicarbonate helps overcome

DIC limitation, but the autotrophic growth of microalgae is ultimately governed by light

availability.

4.3.5. Effects of NaHCO3 Concentration on Growth of C. vulgaris

54

Figure 4.5 Effect of high concentrations of DIC (146, 718, and 1431 mg C/L) and mixing (0

and 125 rpm) on the growth of C. vulgaris.

NaHCO3 was found to be effective in keeping the DIC concentration high, but high

sodium ion concentration may affect the growth of C. vulgaris. Therefore, three different

NaHCO3 concentrations were examined for their effects on growth as shown in Figure 4.5.

Regardless of DIC concentrations, the cultures with mixing (D3-D5) showed faster growth than

those without mixing (D1 and D2). The two high NaHCO3 concentrations of 712 and 1,431 mg

C/L at 125 rpm (D4 and D5) exhibited longer lag phase and slow growth rates compared to the

one with 142 mg C/L (D3). This suggests that a sodium ion concentration should not exceed ~60

mM (i.e. 712 mg C/L = 5,000 mg NaHCO3/L) because a high concentration of sodium ion can

inhibit the growth of the freshwater microalga, C. vulgaris.

4.4. Conclusions

55

In this study, sodium bicarbonate demonstrated its capability as a buffer chemical that

can keep the DIC concentration at a desired level in conjunction with CO2 gas within a pH

window suitable for the autotrophic growth of C. vulgaris. High DIC concentration under

agitation significantly increased the growth rate because suspended cells actively consume DIC

for the photosynthesis reaction. The external mass-transfer resistance was found not to be a rate-

limiting step for the growth. The photosynthesis reaction was found to be limited by DIC

availability at an initial stage of the growth. However, the cell growth was found to be

ultimately limited by light availability due to high microalgal cell density. Further studies about

intracellular DIC mass transfer and photosynthesis reaction for the autotrophic growth of

microalgae will be required for a better mechanistic understanding and reaction kinetic

information.


This study was supported by the College of Engineering and Applied Science at the University

of Cincinnati through faculty start-up funds and the Metropolitan Sewer District of Greater

Cincinnati (MSDGC) under Master Services Agreement No. 85X10431, Task Order No.

0210000209. The authors appreciate their financial support.

4.6. References

[1] H.-P. Luo, M.H. Al-Dahhan, Airlift column photobioreactors for Porphyridium sp. culturing:

Part I. effects of hydrodynamics and reactor geometry, Biotechnology and

Bioengineering, 109 (2012) 932-941.

56

[2] J. Lu, C. Sheahan, P. Fu, Metabolic engineering of algae for fourth generation biofuels

production, Energy and Environmental Science, 4 (2011) 2451-2466.

[3] P.J.l.B. Williams, L.M.L. Laurens, Microalgae as biodiesel and biomass feedstocks: Review

and analysis of the biochemistry, energetics and economics, Energy and Environmental

Science, 3 (2010) 554-590.

[4] S. Zou, Y. Wu, M. Yang, C. Li, J. Tong, Bio-oil production from sub- and supercritical water

liquefaction of microalgae Dunaliella tertiolecta and related properties, Energy and

Environmental Science, 3 (2010) 1073-1078.

[5] M.D. Antezana Zbinden, B.S.M. Sturm, R.D. Nord, W.J. Carey, D. Moore, H. Shinogle, S.M.

Stagg-Williams, Pulsed electric field (PEF) as an intensification pretreatment for greener

solvent lipid extraction from microalgae, Biotechnology and Bioengineering, (2013)

1605-1615.

[6] H. Tang, M. Chen, K.Y. Simon Ng, S.O. Salley, Continuous microalgae cultivation in a

photobioreactor, Biotechnology and Bioengineering, 109 (2012) 2468-2474.

[7] M. Odlare, E. Nehrenheim, V. Ribé, E. Thorin, M. Gavare, M. Grube, Cultivation of algae

with indigenous species – Potentials for regional biofuel production, Applied Energy, 88

(2011) 3280-3285.

[8] L. Amer, B. Adhikari, J. Pellegrino, Technoeconomic analysis of five microalgae-to-biofuels

processes of varying complexity, Bioresource Technology, 102 (2011) 9350-9359.




57

[10] M.K. Lam, K.T. Lee, A.R. Mohamed, Current status and challenges on microalgae-based

carbon capture, International Journal of Greenhouse Gas Control, 10 (2012) 456-469.


1989.



(2009) 13-20.

[13] J.W. Rachlin, M. Farran, Growth response of the green algae Chlorella vulgaris to selective

concentrations of zinc, Water Research, 8 (1974) 575-577.

[14] J.W. Rachlin, A. Grosso, The effects of pH on the growth of Chlorella vulgaris and its

interactions with cadmium toxicity, Archives of environmental contamination and

toxicology, 20 (1991) 505-508.



[16] J. Kim, Z. Liu, J.-Y. Lee, T. Lu, Removal of nitrogen and phosphorus from municipal

wastewater effluent using Chlorella vulgaris and its growth kinetics, Desalination and

Water Treatment, (2013) 1-7.

[17] M. Giordano, J. Beardall, J.A. Raven, CO2 concentrating mechanisms in algae: Mechanisms,

environmental modulation, and evolution., Annual Review of Plant Biology, 56 (2005)

99-131.

[18] J.V. Moroney, A. Somanchi, How do algae concentrate CO2 to increase the efficiency of

photosynthetic carbon fixation?, Plant Physiol., 119 (1999) 9-16.

58

[19] P.L.T. Brian, H.B. Hales, Effects of transpiration and changing diameter on heat and mass

transfer to spheres, AIChE Journal, 15 (1969) 419-425.

[20] S. Chatterjee, G.R. Dickens, G. Bhatnagar, W.G. Chapman, B. Dugan, G.T. Snyder, G.J.

Hirasaki, Pore water sulfate, alkalinity, and carbon isotope profiles in shallow sediment

above marine gas hydrate systems: A numerical modeling perspective, Journal of

Geophysical Research, 116 (2011) B09103.

[21] T. Komada, C.E. Reimers, S.E. Boehme, Dissolved inorganic carbon profiles and fluxes

determined using pH and pCO2 microelectrodes, Limnol. Oceanogr, 43 (1998) 769-781.

[22] L. Yuan-Hui, S. Gregory, Diffusion of ions in sea water and in deep-sea sediments,

Geochimica et cosmochimica acta, 38 (1974) 703-714.

[23] R.E. Zeebe, On the molecular diffusion coefficients of dissolved, and and their dependence

on isotopic mass, Geochimica et Cosmochimica Acta, 75 (2011) 2483-2498.

[24] M. Bertrand, D. Parmentier, O. Lebaigue, E. Plasari, F. Ducros, Mixing Study in an

Unbaffled Stirred Precipitator Using LES Modelling, International Journal of Chemical

Engineering, 2012 (2012).

[25] R.B. Bird, W.E. Stewart, E.N. Lightfoot, Transport phenomena, Wiley. com, 2007.

[26] S. Nagata, K. Yamamoto, K. Hashimoto, Y. Naruse, Flow patterns of liquids in a cylindrical

mixing vessel with baffles, Memoirs of the Faculty of Engineering, Kyoto University, 21

(1959) 260.

[27] S. Nagata, K. Yamamoto, M. Ujihara, Flow patterns of liquid in a cylindrical mixing vessel

without baffles, Memoirs of the Faculty of Engineering, Kyoto University, 20 (1958)

336-349.

59

[28] E. Evers, A model for light-limited continuous cultures: Growth, shading, and maintenance,

Biotechnology and Bioengineering, 38 (1991) 254-259.

[29] I.S. Suh, S.B. Lee, A light distribution model for an internally radiating photobioreactor,


[30] Y.-S. Yun, J. Park, Attenuation of monochromatic and polychromatic lights in Chlorella

vulgaris suspensions, Applied Microbiology and Biotechnology, 55 (2001) 765-770.

60

Growth Modeling of Chlorella vulgaris with regard to Chapter 5

Photolimitation and Photoinhibition effects

5.1. Introduction

The interest in microalgae has been increasing as a biofuel feedstock due to their

substantial amount of lipids (e.g., 15-50% dry cell density) [1]. In addition, autotrophic growth

of microalgae was considered as an effective way of reducing CO2, a major contributor of global

warming [2-5]. Also the growth rate of microalgae is faster than terrestrial plants due to their

high rate of photosynthesis and high efficiency of solar energy utilization (up to 10 %) [6-8].

Light is one of the most important parameters determining the microalgae photosynthesis

rate as per the following reaction (1).

2 2CO nutrients light O Biomass (1)

In an earlier study, a growth model was established by using overall specific growth rate with

respect to incident light intensity [9-11]. However, incident light intensity is attenuated inside

the reactor since the light is absorbed or scattered by the algal cell, known as a shading effect

[12]. In this respect, several studies were employed to describe the light attenuation inside the

reactor associated with the shading effect, known as the light distribution model [11, 13, 14]. In

the studies, maximum specific growth rate with respect to average light intensity was used under

low cell density. However, the specific growth rate changes with respect to time. It has been

reported that the rate at the same average light intensity can also decrease under high light

intensity because high light inhibits the growth rate of microalgae, known as a photoinhibition

effect [15-18]. Thus, those two factors should be considered to establish the growth model.

However, no growth model coupling these two factors has been established.

61

In this study, we established a novel model accounting for the effects of photolimitation

and photoinhibtion. The validity of the model was confirmed by comparing the experimental

data and simulated data depending on light intensity on the surface of the reactor and reactor

size.


5.2.1. Organism and Cultivation Conditions

The C. vulgaris used (UTEX #2714) in this study was obtained from UTEX at the

University of Texas at Austin. The culture medium was prepared by following the modified

Shuisheng-4 medium [19]. However, 1000 mg/L of sodium bicarbonate (NaHCO3) in this study

was used for the medium because the concentration was determined to be optimal for the culture

of C. vulgaris according to our previous study [20]. Then, the 1,800 mL of the prepared medium

was added to a 2-L cylindrical reactor (21 cm (height) × 11 cm (diameter)).

The cultures were grown in different light intensities (0, 30, 55, 80, 197, 476, and 848

µmol∙m-2

∙s-1

) on the surface of the reactor with fluorescent lamps with 6,500 K color temperature

and a 16-h light and 8-h dark cycle was applied to the culture. The light intensity was measured

using a light quantum meter (LightScout Foot-Candle meter, Spectrum Technologies, Inc).

During the culture, the mediums were mixed at 125 rpm because the mixing speed was found to

be enough for homogenizing the algal cell in the reactor according our previous study, and the

pH of the media was controlled at 7 by adding CO2(g) once daily to maintain an optimal pH

range (pH=7-8) for C. vulgaris, as reported by Kim et al. [20-22]. In addition, essential nutrients

were sufficiently provided by monitoring dissolved inorganic carbon, nitrogen and phosphorus

concentrations.

62

5.2.2. Analytical Methods

The cell density of C. vulgaris was measured using a UV-vis spectrophotometer (UV-

1800, Shimadzu Scientific Instruments) as described by Kim et al. [23, 24]. The absorption

coefficient (Ka) of the C. vulgaris (0.347±0.014 m2/g) was determined as described by Molina

Grima et al. [14].

Growth modeling was carried out using COMSOL 4.3a software package and a

regression analysis was performed by OriginPro 8.0 software package.

5.2.3. Model Description

The cell growth per unit time for the batch reactor can be determined by using equation

(2) as follows:

dW

Wdt

(2)

where W is the biomass concentration (mg/L), t is time (h), and is specific growth rate (, h-1

)

[25]. In equation (2), it can be assumed that the specific growth rate is dependent on light

intensity because the experiment was employed under nutrients-sufficient, optimal pH and

mixing conditions during the culture. In general, light intensity affects the specific growth rate in

two ways: photolimitation and photoihibition [16]. Photolimitation occurs due to increasing the

biomass concentration during the growth phase. During the growth phase, the light intensity

gradually attenuates as the biomass concentration increases, due to the shading effect of biomass

and thus the specific growth rate decreased with respect to time, as a result of a decrease in the

photon flux [11-14]. Photoinhibition is related to Iin. The specific growth rate can be inhibited

by the high light intensity, as was reported, the high light intensity reduces the photosynthesis

63

rate of microalgae [16, 18]. Therefore, our model was developed by adding photolimitation and

photoinhibition effects on the growth model.

5.2.3.1. Light Distribution Model: Photolimitation with respect to Biomass

Concentration

The scheme of the light distribution model with respect to the shading effect of biomass

is shown in Figure 5.2(a), which is derived from Lambert-Beer law as described by Ever et al [13,

14]. According to the model, the light intensity can be determined by using equation (3).

2 2 2 0.5

0 0 00

( , ) exp{ [( )cos ( ( ) sin ) ]}inII r W W R r R R r d

(3)

where Iin is the light intensity (µmol∙m-2

∙s-1

) on the surface of the reactor, R is the radius (m) of

the reactor, r0 is the light path, θ (radian) (0≦θ≦π) is the angle of the light path with the dashed

line passing through the center, α is the absorption coefficient (m2∙g

-1) and I is a function of

position and initial biomass concentration [13, 14]. The average light intensity ( ( )I W ) that is

dependent on biomass concentration can be determined by using equation (4):

2 2 2 0.5

0 0 00 0

( ) exp{ [( )cos ( ( ) sin ) ]}r

inII W W R r R R r d dr

r

(4)

where ( )I W is a function of initial biomass concentration [13, 14]. We can add the

photolimitation effect on our model by using those two light intensities (I, ( )I W ).

5.2.3.2. Specific Growth Model with respect to Iin associated with the Photoinhibtion

Specific growth rate can be determined by using equation (5):

1 2 1

2 1

ln( / )( )

W Wh

t t

(5)

64

where the specific growth (1( )h

) was defined as the increase in biomass (W, mg/L) per unit

time (t, h). In this study, the specific growth rate changed over time due to the photolimitation

effect. Therefore, it is necessary to determine an expression which correlates the specific growth

rate with photolimitation effect. Accordingly, Molina proposed the following correlation

between the specific growth rate and average light intensity, and accounts for photolimitation

effect, shown in equation (6).

max

n

n n

k

I

I I

(6)

where µ is the specific growth rate (, h-1

), µmax is the maximum specific growth rate, I is

average light intensity, kI is saturation concentrate and n fitting parameter. Here, the parameter

can be determined by non-linear regress analysis.

However, the specific growth rate at the same average light intensity was dependent on

the light intensity on the surface of the reactor due to the photoinhibition effect of high light and

thus the specific growth rate expression also differs depending on the light intensity at the

surface of the reactor. Therefore, different specific growth rate expression is dependent on the

light intensity on the surface of the reactor should be used to precisely simulate growth of

microalgae. We can add the photoinhibition effect on our model by using different specific

growth rate expressions that are dependent on light intensity on the surface of the reactor.

Finally, we can simulates the growth of microalgae with respect to Iin, reactor size and initial cell

density by coupling with equations (2), (3), (4), and (6).


65

5.3.1. Growth of C. vulgaris in Different Light Intensities on the Surface of Reactor

Figure 5.1 (a) Growth of C. vulgaris with respect to time dependent on light intensity (0, 30,

55, 80, 197, 476, and 848 µmol∙m-2

∙s-1

) on the surface of reactor, (b) Specific growth rate (h-

1) with respect to time dependent on light intensity, and (c) Specific growth rate (h

-1) with

respect to biomass concentration.

Figure 5.1(a) shows the growth kinetics of C. vulgaris under different light intensity (0,

30, 55, 80, 197, 476, and 848 µmol∙m-2

∙s-1

) on the surface of the reactor. The overall growth

kinetics of C. vulgaris with respect to time decreased because the increased biomass

66

concentration may have attenuated the light distribution inside reactor by shading. This shading

effect of biomass was widely reported in previous studies [11, 13, 14]. Thus, the specific growth

rate with respect to time and biomass concentration was plotted using equation (5) as shown in

Figure 5.1(b) and 1(c), respectively. As a result the overall specific growth rate exponentially

decreased with respect to time and biomass concentration. The results indicate that the light-

limitation effect derived from the increase of biomass with respect to time during the growth

phase should be considered in order to precisely develop a model.

In addition, the growth rate of C. vulgaris increased with the increase of light intensity on

the surface of the reactor, but the rate did not increase furthermore beyond Iin=197 µmol∙m-2

∙s-1

because the accelerated photosynthesis rate derived from the increase of photon flux might be

neutralized by the photoihibition effect derived from the high light intensity, which was widely

reported, shown in Figure 5.1(a) [16-18]. The result indicates that the photoinhibition effect also

should be considered to develop the model along with the photolimitation effect.

5.3.2. Light Distribution Model with respect to Biomass Concentration

67

Figure 5.2 (a) Schematic representation of light distribution model, and (b) simulation vs.

experimental data of the light distribution with respect to the r0 inside reactor.

Figure 5.2(b) shows the light attenuation with respect to the light path (r0) and biomass

concentration determined by using equation (3) and comparison between the simulated data and

experiment data under Iin=80 µmol∙m-2

∙s-1

and r=5.5 cm, the overall light intensity decreased

with the increase of light path because the produced photon flux was absorbed by biomass. Also,

the degree of light intensity loss increased with the increase of the biomass concentration due to

the shading effect. In addition, it was observed that the simulated data matched well with the

experimental data. Therefore, the result indicates that we can use the light distribution model to

develop our growth model.

5.3.3. Specific Growth Rate with respect to Light Intensity

68

Figure 5.3 Specific growth rate with respect to average light intensity depending on light

intensity on the surface of reactor.

Figure 5.3 shows the specific growth rate with respect to the average light intensity. Here,

the average light intensity was determined by using equation (4) and then, the parameters of the

specific growth rate expression were independently determined depending on the light intensity

on the surface of the reactor by the non-linear regression fit of the experimental data in Figure

5.3 and summarized in Table 5.1. The rise of the specific growth rate curve with the increase of

the light intensity on the surface of the reactor decreased due to the photoinhibition effect of the

high light intensity, which is widely reported [16-18]. Therefore, the result indicates that

different specific growth rate expressions should be used dependent on a given light intensity on

surface of the reactor.

Table 5.1 Parameters of specific growth rate dependent on the light intensity on the surface

of reactor.

Light intensity

on the surface of

reactor

(mol∙m-2

∙s-1

)

Parameters

max (h-1

) Ik ((mol∙m-2

∙s-1

) n R2

30 0.1718 29.54 1.04 0.998

55 0.0824 10.26 1.39 0.995

80 0.0918 18.11 1.30 0.994

197 0.0928 34.04 1.23 0.993

476 0.0817 61.10 1.39 0.994

848 0.0928 146.52 1.23 0.993

69

5.3.4. Growth Model and Simulation

Figure 5.4 (a) Simulated a specific growth rate with respect to time, (b) light intensity with

respect to the light path and time, (c) average light intensity with respect to time, and (d)

growth of C. vulgaris with respect to time.

Figure 5.4 shows the growth simulation results of C. vulgaris under the condition of 80

µmol∙m-2

∙s-1

light intensity on the surface of the reactor and 11 cm of reactor diameter. The

specific growth rate sharply decreased with respect to time (Figure 5.4(a)) associated with the

increase of biomass concentration. The biomass concentration gradually increased with time

70

(Figure 5.4(d)). In addition, the light intensity along with the increase of light path or time was

decreased due to the attenuation of light intensity associated with the shading effect of biomass

(Figure 5.4(b)) and the average light intensity decreased with time due to the increase of biomass

concentration (Figure 5.4(c)). These results indicate that we can simulate specific growth rate,

biomass concentration with respect to time, average light intensity, and light intensity with

respect to time and location by using our model. The simulation was compared to the

experimental data in order to validate our model as described in the following section.

5.3.5. Experimental Data vs. Growth Modeling

Figure 5.5 Comparison between experimental data and simulated data (a) growth kinetics

of C. vulgaris with respect to time depending on light intensity on the surface of reactor,

and (b) growth kinetics of C. vulgaris with respect to time depending on the reactor size.

71

The comparison between the simulation and experimental data based on the light

intensity and the size of reactor is shown in Figure 5.5. According to the simulated growth of C.

vulgaris, the growth rate increased depending on the light intensity on the surface of the reactor

and did not increase furthermore beyond Iin=197 µmol∙m-2

∙s-1

due to the photoihibition effect.

The growth rate of C. vulgaris decreased with the increase of the reactor size because average

light intensity increased owing to the decrease of light path. The overall growth patterns are

dependent on light intensity on the surface of the reactor and reactor size using simulation were

well in agreement with the experimental data. Therefore, it can be concluded that we can

precisely simulate the growth of C. vulgaris based on light intensity on the surface of the reactor

and the size of reactor.

5.4. Conclusions

The growth model of C. vulgaris with respect to photolimitation and photoinhibtion was

proposed in this study. This study can be summarized as follows:

1. The growth rate of C. vulgaris is influenced by photolimiation and photoinhibition effects

and thus those two factors should be considered in order to establishing a growth model.

2. The proposed model successfully simulated the growth profiles depending on the light

intensity and the reactor size and was found to be well matched with the experimental data.

3. The model would be applicable to estimate the growth of C. vulgaris for any type of

continuous photobioreactor by incorporation of the model into the performance equation of the

photobioreactor.

72

5.5. Acknowledgements

This study was supported by the College of Engineering and Applied Science at the

University of Cincinnati through faculty start-up funds and the Metropolitan Sewer District of

Greater Cincinnati (MSDGC) under Master Services Agreement No. 85X10431, Task Order No.

0210000209. The authors appreciate their financial support.

5.6. References



Science, 3 (2010) 554-590.

[2] J.R. Benemann, CO2 mitigation with microalgae systems, Energy Conversion and

Management, 38, Supplement (1997) S475-S479.

[3] Y.-C. Jeon, C.-W. Cho, Y.-S. Yun, Measurement of microalgal photosynthetic activity

depending on light intensity and quality, Biochemical Engineering Journal, 27 (2005)

127-131.

[4] J. Keffer, G. Kleinheinz, Use of Chlorella vulgaris for CO2 mitigation in a photobioreactor,

Journal of Industrial Microbiology and Biotechnology, 29 (2002) 275-280.

[5] B. Wang, Y. Li, N. Wu, C.Q. Lan, CO2 bio-mitigation using microalgae, Applied

Microbiology and Biotechnology, 79 (2008) 707-718.

[6] Y. Chisti, Biodiesel from microalgae., Biotechnology Advances, 25 (2007).




73

[8] G.W. Huber, S. Iborra, A. Corma, Synthesis of transportation fuels from biomass: chemistry,

catalysts, and engineering., Chemical Reviews, 106 (2006) 4404-4498.

[9] T. Ogawa, H. Kozasa, G. Terui, Studies on the growth of Spirulina platensis (II) growth

kinetics of an autotrophic culture, Journal of Fermentation Technology, 50 (1971) 143-

149.

[10] H. Qi, G.L. Rorrer, Photolithotrophic cultivation of Laminaria saccharina gametophyte

cells in a stirred-tank bioreactor, Biotechnology and Bioengineering, 45 (1995) 251-260.

[11] I.S. Suh, S.B. Lee, A light distribution model for an internally radiating photobioreactor,


[12] Y.-S. Yun, J. Park, Attenuation of monochromatic and polychromatic lights in Chlorella

vulgaris suspensions, Applied Microbiology and Biotechnology, 55 (2001) 765-770.

[13] E. Evers, A model for light-limited continuous cultures: Growth, shading, and maintenance,


[14] E.M. Grima, F.G. Camacho, J.A.S. Pérez, J.M.F. Sevilla, F.G.A. Fernández, A.C. Gómez, A

mathematical model of microalgal growth in light-limited chemostat culture, J. Chem.

Technol. Biotechnol., 61 (1994) 167-173.

[15] E.M. Grima, F.G. Camacho, J.S. Pérez, F.A. Fernández, J.F. Sevilla, Growth yield

determination in a chemostat culture of the marine microalgaIsochrysis galbana, Journal

of Applied Phycology, 8 (1996) 529-534.

[16] E.M. Grima, J. Sevilla, J. Pérez, F.G. Camacho, A study on simultaneous photolimitation

and photoinhibition in dense microalgal cultures taking into account incident and

averaged irradiances, J. Biotechnol., 45 (1996) 59-69.

74

[17] J. Sandnes, T. Källqvist, D. Wenner, H.R. Gislerød, Combined influence of light and

temperature on growth rates of Nannochloropsis oceanica: linking cellular responses to

large-scale biomass production, Journal of Applied Phycology, 17 (2005) 515-525.

[18] A. Vonshak, G. Torzillo, L. Tomaseli, Use of chlorophyll fluorescence to estimate the effect

of photoinhibition in outdoor cultures of Spirulina platensis, Journal of Applied

Phycology, 6 (1994) 31-34.


1989.








[23] J. Kim, B.P. Lingaraju, R. Rheaume, J.-Y. Lee, K.F. Siddiqui, Removal of ammonia from

wastewater effluent by Chlorella vulgaris, Tsinghua Science & Technology, 15

(2010) 391-396.




75

[25] A. Shotipruk, P.B. Kaufman, H.Y. Wang, Conceptual Design of LED-Based Hydroponic

Photobioreactor for High-Density Plant Cultivation, Biotechnology progress, 15 (1999)

1058-1064.

76

Effects of Dissolved Inorganic Carbon Concentrations, pH and Chapter 6

Harvest Time on Lipid Accumulation of Green Microalga Neochloris

oleoabundans in Two-stage Cultivation System

6.1. Introduction

CO2 is a major contributor to global warming and the interest of CO2 reduction has

recently increased [1, 2]. Microalgae are able to utilize CO2 as an inorganic carbon source for

autotrophic growth [3, 4]. In addition, microalgae can grow faster than terrestrial plants due to

their high photosynthesis rates and also produce a large amount of lipids (about 15-50% dry cell

weight) [5-7]. In this sense, the attention to microalgae has been increased as a renewable

biomass feedstock [8, 9].

Open ponds for cultivating microalgae are a favorable system in the economics view of

scale-up because the cost of open systems is approximately 4 times lower than closed systems

[10]. However, the growth rate of microalgae in open pond systems is fairly slow by autotrophic

growth using CO2(g) due to the low production of DIC (= -

3 2HCO +CO (aq) ) by CO2(g) as a

result of the low solubility of CO2(g) under atmospheric pressure. Thus, closed systems are

widely used for cultivating microalgae using CO2(g) in order to increase DIC production by

increasing a pCO2 [11, 12]. In this respect, Kim et al. recently suggested NaHCO3 based system

using a used sorbent after absorbing CO2(g) using sodium carbonate in order to increase the

production of DIC under atmospheric pressure [13]. The system can simply increase DIC

concentration under open systems. Furthermore, it was reported that the increased DIC

concentration promoted the photosynthesis rate of green alga C. vulgaris. However, the

NaHCO3 based system was not applied to other green microalgae. In this respect, it is necessary

77

to evaluate whether the system can be employed to other green microalgae or not as well as C.

vulgaris.

In general, microalgae grow well under nutrient-sufficient condition, but the lipids do not

accumulate under the condition because most of absorbed DIC in microalgal cell are fixed as a

form of protein, carbohydrate and pigment rather than lipids [14]. In contrast, it was widely

reported that lipids in microalgal cell were accumulated under stressful environments such as

nitrogen deprivation and low temperature conditions [15-17]. However, the growth of

microalgae in those conditions is strongly restricted. In this respect, a two-stage cultivation

process was proposed to increase total lipid productivity by separating two stages: (1) the first

stage phase: the growth phase under nutrient-sufficient condition to maximize biomass

productivity, and (2) the second-stage phase under stressful conditions, particularly nitrogen

deprivation because the method is reported as the most effective method for lipid accumulation:

the lipid accumulation phase to maximize lipid accumulation [14, 18]. In fact, Go et al and Su et

al. can increase the total lipid productivity approximately 1.5-2.8 times than single-stage process

by using two-stage cultivation process [19, 20].

For the second-stage phase, nitrogen deprivation condition is generally used but other

detailed conditions such as DIC, pH, and harvest time are not described before. DIC may be one

of the important factors on the lipid accumulation because bicarbonate ion is used as a carbon

source in the first step of fatty acids synthesis from acetyl-CoA to Malonly-CoA for the

regulation of lipid biosynthesis [21]. pH may be an important factor because many of the lipid

metabolism of microalgae were governed by pH [22-24]. Lastly, the point of harvesting

microalgae may also be an important factor because the dying algal cell may not be unable to

78

maintain lipid content in algal cell during death. Thus, it is necessary to confirm the effect of

DIC, pH and harvesting time on the lipid accumulation under nitrogen deprivation condition.

In this study, a NaHCO3 based system was used to control DIC concentration for both

the first-stage phase and the second-stage phase in open systems. The objectives of this study

were to examine the effect of DIC concentration for the growth rate of green microalgae, N.

oleoabundans during the first-stage phase and the effect of DIC, pH and harvesting time for the

lipid accumulation in microalga cell during the second-stage phase.


6.2.1. Culture Medium and Conditions

N. oleoabundans used (UTEX #1185) in this study was obtained from UTEX at the

University of Texas at Austin. The cultivation was performed by dividing into two stages: first-

stage under nutrients-sufficient condition and second-stage under nitrogen-deprivation condition.

For the first-stage phase, a culture medium was prepared by following the modified Shuisheng-4

medium and 1800 mL of the prepared medium was added to a 2-L clear standard bottle (Thermo

Fisher Scientific Inc.) [25]. In particular, sodium nitrate (NaNO3) in this study was used instead

of ammonium sulfate ((NH4)2SO4) as a nitrogen source because Li et al. reported that nitrate

showed the maximum biomass concentration compared to the use of ammonium and urea [26].

850 mg of NaNO3/L (10 mM) of sodium nitrate was used for the first-stage culture because Li et

al. reported that the concentration showed maximum biomass productivity for N. oleoabundans

[26]. NaHCO3 was not added to the medium during this step. Then, an initial DIC concentration

in a culture medium was controlled by a different amount of NaHCO3: 100 mg of NaHCO3/L

(0.01 M) for F1, 252 mg of NaHCO3/L (0.003 M) for F2, 420 mg of NaHCO3/L (0.005 M) for

79

F3, 840 mg of NaHCO3/L (0.01 M) for F4, 2,520 mg of NaHCO3/L (0.03 M) for F5, 4,200 mg of

NaHCO3/L (0.05 M) for F6, 8,401 mg of NaHCO3/L (0.1 M) for F7, 25,202 mg of NaHCO3/L

(0.3 M) for F8, and 42,004 mg of NaHCO3/L (0.5 M) for F9 culture for first stage experiment.

Then, CO2(g) was added to all the culture media to control the initial pH of the media at 7 and

this pH control was repeated once daily. During the culture, F1, F2, F3, F4, F5, F6, F7, F8, and

F9 culture media were mixed by using a magnetic stirrer at a speed of 350 rpm. The same

amount of extra nutrienta in Shuisheng-4 medium including nitrogen and phosphorus was added

to all media every 5 days except for NaHCO3 to ensure a sufficient supply of essential nutrients

and those nutrients were monitored during the culture.

For the second-stage phase, initially the large amount of N. oleoabundans was cultivated

in eight of 1800 mL of the prepared mediums in 2-L clear standard bottle. The medium was

prepared by the same method with above first-stage phase and 10 mM of NaNO3 and 0.01 M of

NaHCO3 were used and other nutrient mediums for the culture. After the culture, the biomass

was centrifuged using Thermo Scientific Sorvall Primo Benchtop Centrifuge with 3500 rpm.

The harvested biomass washed 3 times by Deionized water in order to remove residual nutrients

and used for the second-stage phase. Then, for the second-stage phase, 1800 mL of prepared

medium by following the modified Shuisheg-4 medium was added into 2-L of bottle but NaNO3

was not added into the medium in order to making nitrogen-deprivation condition. NaHCO3 was

not added to the medium during this step. Then, an initial DIC concentration in a culture

medium was controlled by a different amount of NaHCO3: 0 mg of NaHCO3/L (0 M) for S1 and

S2, 252 mg of NaHCO3/L (0.003 M) for S3, S4, S5, and S6, 2,520 mg of NaHCO3/L (0.03 M)

for S7, S8, S9, and S10 for second stage experiment. Then, CO2(g) was added to S5, S6, S9 and

S10 to control the initial pH of the media at 7 and this pH control was repeated once daily.

80

During the culture, S1, S2, S3, S4, S5, S6, S7, S8, S9, and S10 culture media were mixed by

using a magnetic stirrer at a speed of 350 rpm. Then, it was ensured that the nitrogen-

deprivation in all medium was confirmed by using Dionex Ion-chromatography (LC20

chromatography enclosure-GP50 Gradient pump-CD25 conductivity detector with IonPacR

AS14 analytical column (4 × 250 mm)).

During the culture, fluorescent lamps with 6500-K color temperature similar to natural

sunlight color temperature were used as a light source. The incident light intensity at the reactor

surface was set to 353.75±9.28 µmol/(m2 s)), and a 16-h light and 8-h dark cycle was applied to

all the cultures. All the culture media were open to an atmosphere at 25 ºC.

6.2.2. Determination of Cell Density of N. oleoabundans

The cell density of N. oleoabundans was determined by measuring the optical density at

750 nm for every 24 hours by using a UV-vis spectrophotometer (UV-1800, Shimadzu Scientific

Instruments) because the wavelength do not interfered by pigment according to Griffiths et al

[27-29]. The absorbance of UV spectrophotometer at 750 nm was calibrated by measuring the

weight of dried N. oleoabundans. Then, the weight of dried biomass was obtained from the

prepared calibration curve.

6.2.3. Determination of DIC, NO32-

, and PO43-

Concentration

An acid-base titration method was used to determine the concentrations of DIC species

present in the aqueous phase [13, 30, 31]. This titration method determines a TIC concentration

(- 2-

3 3 2 3 2=[HCO ]+[CO ]+[H CO ]+[CO (aq)] ) in a 15-mL sample using 0.1 N and 0.01 N HCl

solutions for the titration of high and low carbon concentrations, respectively. Then, the DIC

81

concentrations were calculated by using the equilibrium relations shown in the next section

among HCO3-, CO3

2-, H2CO3, and CO2(aq).

The concentration of NO32-

was determined using Ion-chromatography and PO43-

was

determined using Phosver 3 phosphate regent (Hach company).

6.2.4. Determination of Total Lipids by Gravimetric Method

6.2.4.1. Collection of Algae Biomass

Algae biomass was collected using a bench centrifuge (Type 2010, Thermo-fisher, US) at

3,500 rpm for 10 minutes. Then the biomass was dried in a freeze dryer below -40 ºC and 0.133

mbar for 3 days (model: Labconco Freezone 12 Freeze Dryer).

6.2.4.2. Solvent Extraction of Total Lipids

Total lipids were extracted using solvent extraction modified from the Bligh & Dyer’s

method [32]. Lipids extraction was conducted using 400 mg of dried algae and 30 mL of a

solvent mixture (chloroform:methanol=2:1) for 24 hrs at room temperature. Then the liquid was

centrifuged at 4,000 rpm for 10 min to remove algae debris followed by filtration using a 0.45

µm filter (Type HA, Millipore, US) to further remove all suspended solid residues. 10 mL of

deionized water was added to the filtrate and the filtrate solution was shaken vigorously for 2

min. Aqueous and organic phases were separated by centrifugation at 3,500 rpm for 5 min. The

bottom phase (chloroform) was recovered and this washing step was repeated two more times to

remove any residual methanol. To determine the weight of total lipids, the chloroform phase

was transferred to a pre-weighed flask and the solvent was evaporated completely using a

82

rotatory evaporator (Model: R-II, Buchi, Switzerland) at 40 ºC. The weight of the flask was

measured again to measure the total lipid content in the sample.

Figure 6.1 Effects of different DIC concentrations on the growth of N. oleoabundans under

nutrient-sufficient condition during first-stage phase: (a) initial DIC of 0.001 M, 0.003 M,

0.005 M, 0.01 M, 0.03 M, 0.05 M, 0.1 M, 0.3 M, and 0.5 M of NaHCO3/L under 350 rpm

mixing speeds. Note: CO2(g) was added all culture media to control the pH of the media at

7 once daily; (b) nitrogen concentration at 312 h under different DIC concentrations; (c)

83

phosphorus concentration at 312 h under different DIC concentrations; (d) pH change

under different DIC concentrations.

6.2.4.3. Separation of Neutral Lipids and Polar Lipids

Neutral and polar lipids were separated by column chromatography using silica gel (70-

230 mesh, Alfa Aesar, US). For every 200 mg of total lipids, 15 g of silica gel and 170 mL of

eluent solution (chloroform) were used for neutral lipids and 15 g of silica gel and 170 mL of

eluent solution (methanol) were used [33].


6.3.1. The first-Stage Phase: Growth of N. oleoabundans under Different DIC

Concentration under Nutrient-Sufficient Condition

N. oleoabundans for the first-stage phase was cultivated in nine different initial DIC

concentrations of 0.001, 0.003, 0.005, 0.01, 0.03, 0.05, 0.1, 0.3, and 0.5 M of DIC using

NaHCO3 under mixing in open system. During the culture, the pH values of the culture media

were increased up to pH=11.9 as a result of OH- ion production by N. oleoabundans, but the

fluctuation of pH was decreased with respect to the increase of initial NaHCO3 concentrations in

culture medium because the used NaHCO3 plays the role of buffer to maintain pH as shown in

Figure 6.1(d). Thus, the increased pH values were controlled at 7 by adding CO2(g) once daily.

Essential nutrients except for DIC were also monitored and kept by adding the same amount of

extra nutrients to all media every 5 days during the growth phase as shown in Figure 6.1(b) and

(c).

84

The growth in F1-F5 cultures gradually increased after 72 hrs to adapt to those given

condition as a lag phase. The growth rate in those cultures was noticeably increased with respect

to the increase of the initial DIC concentration (from 0.001 M to 0.03 M) in culture medium

which showed similar trend of DIC effect on autotrophic growth of C. vulgaris which was

previously reported. Otherwise, the growth in F6 and F7 cultures gradually increased after

having slightly longer lag phase (96 hrs) than that in F1-F5 cultures because a slightly high

sodium ion concentration in F6 and F7 mediums might inhibit the growth of N. oleoabundans

even if the DIC concentration in those mediums was higher than that in F5 medium. However,

the biomass concentration in F6 and F7 cultures became higher than that in F5 medium after 192

hrs due to the high growth rate in those cultures after passing the lag phase derived from a high

DIC. In addition, the biomass concentration in F7 culture became a higher level than the other

cultures due to the substantially high growth rate. The growth in F8 and F9 cultures gradually

increased after having considerably longer lag phase (144 hrs for F8 culture and 240 hrs for F9

culture, respectively) because the extremely high sodium ion concentration strongly inhibits the

growth of N. oleoabundans. This inhibition effect of sodium ion for N. oleoabundans is

analogous tendency with that for C. vulgaris which was previously reported. Otherwise, the

tolerance (approximately 100 mM) of N. oleoabundans was approximately 1.7 times higher than

that (60 mM) of C. vulgaris which was previously reported [13]. Therefore, the positive effect

of DIC and inhibition effect of high concentration of sodium ion on the autotrophic growth of N.

oleoabundans were reconfirmed similar to the growth of C. vulgaris based on NaHCO3 in open

systems.

6.3.2. First-Stage Phase: Lipid Accumulation in N. oleoabundans under Different DIC

85

Concentration for the First-Stage Phase


method in different DIC concentrations under nutrient-sufficient condition.

The resultant biomass after the first-stage phase was harvested at 312 hrs and dried in

Freeze dryer for 3 days. Then, the neutral and polar lipids in the dried biomass were determined

by gravimetric method as shown in Figure 6.2. Total lipids for F1-F4 cultures were slightly

increased with respect to the increase of initial DIC concentration from 13.7% to 21.2%. When

the initial DIC concentration is higher than 0.01 M, however, the total lipids for F4-F8 cultures

did not show a noticeable difference with respect to the change of initial DIC concentration with

having a small standard deviation (±1.6%). In addition, the total lipids in F9 culture was the

lowest (12.4%) even if the DIC concentration was the highest than any other cultures because the

86

extremely high concentration of DIC might inhibit the total lipids accumulation in N.

oleoabundans during the growth. However, overall total lipids change dependent on initial DIC

concentrations did not show a noticeable difference with having a small standard deviation

(±3.0%). In addition, the neutral lipids in all cultures were insignificant (less than 5%: 1.71% for

F1, 1.49 % for F3, 4.27 % for F3, 3.5% for F5, 1.49% for F6, 3.51 % for F7, 1.7 % for F8, 4.99 %

for F9) and the change also did not show a noticeable difference with having small standard

deviation (±1.4%). Therefore, it was noted that the different concentration of DIC did not

efficiently effect on the total and neutral lipids content in N. oleoabundans under nutrient-

sufficient condition.

87

6.3.3. Second-Stage Phase: Growth of N. oleoabundans under Different DIC Concentration

under Nitrogen-Deprivation Condition

Figure 6.3 Effects of different DIC concentrations, existence of pH control, and harvest

time on the growth of N. oleoabundans under nitrogen-deprivation condition during

second-stage phase: (a) initial DIC of 0.003 M and 0.01 M of NaHCO3/L under 350 rpm

mixing speeds. Note: CO2(g) was added S5, S6, S9, and S10 culture media to control the pH

88

of the media at 7 once daily; (b) pH change in different DIC concentrations, existence of pH

control.

N. oleoabundans for the second-stage phase was cultivated in there different

concentrations of initial DIC of 0 M (S1 and S2), 0.003 M (S3, S4, S5 and S6) and 0,03 M (S7,

S8, S9, and S10) using NaHCO3 under mixing in open system as shown in Figure 3(a).

According to the equilibrium relationship among inorganic carbon species (CO2(aq), HCO3- and

CO32-

), DIC concentration (CO2(aq) and HCO3-), which is essential inorganic carbon source for

lipid biosynthesis, was maximized in the pH range of 7-8.3. On the other hand, CO32-

became

most abundance inorganic carbon specie when pH is higher than 8.3. Thus, it was postulated

that pH control to 7 for second-stage phase may help the lipid accumulation mechanism in

microalgae. In this sense, it is necessary to assess the effect of pH on the lipid accumulation of N.

oleoabundans. Thus, the pH of two of cultures having the same initial DIC concentration (0.003

M for S3 and S4 and 0.03 M for S7 and S8) was not controlled and the other of them (0.003 M

for S5 and S6 and 0.03 M for S7 and S8) was controlled to pH 7. However, the pH of the

cultures having no DIC (0 M for S1 and S2) was not controlled because the pH was not increased

during the culture because the microalgae did not grow derived from the DIC limitation together

with nitrogen deprivation as shown in Figure 6.3(b). In addition, it is necessary to determine

optimum harvest time for the second-stage phase because the increased lipids by nitrogen

deprivation obviously may be decreased at certain point owing to the metabolism change of lipid

accumulation accompanied by DIC concentration and pH control. In this respect, one of the

culture (S1, S3, S5, S7, and S9) having the same initial DIC and pH control condition was

harvested at stationary phase and the other of culture was harvested at death phase (S6, S8, and

89

S10). However, the growth curves of S2 and S4 cultures did not reach to death phase and thus

the biomass in those cultures was harvested at 432 hrs.

The growth in S1 and S2 cultures was not observed due to the limitation of both the DIC

and nitrogen as shown in Figure 6.3(a). On the other hand, the growth in S3-S10 cultures, when

the DIC existed in medium, was observed even though nitrogen was deprived in those cultures

because the DIC might help to overcome the growth limitation derived from the nitrogen

deprivation. With respect to the effect of the DIC concentration on the growth, the biomass

concentration (~500 mg/L) with a high DIC was a slightly higher level than that (~450 mg/L)

with a low DIC because a high DIC might promote the growth rate even though nitrogen was

deprived in those cultures and the inclination is analogous with the trend under the nutrient-

sufficient condition. With respect to effect of pH control on the growth, the growth phase

without pH control slowly moved to stationary phase, and also slowly moved to death phase or

even did not reach to the phase at 432 hrs for S2 and S4 cultures. However, the growth phase

with pH control rapidly moved to stationary phase (96 hrs for S5, S6, S9, and S10 cultures) and

the stationary phase also rapidly moved to death phase because the pH control might help to

rapidly maximize the biomass concentration under nitrogen deprivation condition. Therefore, it

was noted that the culture with a high DIC under pH control was considered as the optimal

condition to maximize the biomass concentration and be shorten the second-stage phase.

90

6.3.4. Second-Stage Phase: Lipid Accumulation in N. oleoabundans under Different DIC

Concentration under Nitrogen-Deprivation Condition


method in different DIC concentrations under nitrogen-deprivation condition during

second-stage phase.

The resultant biomass for the second-stage phase was harvested at stationary phase (S1,

S3, S5, S7, and S9) and death phase (S6, S8, and S9), respectively. However, S2 and S4 cultures

were harvested at 432 hrs because the growth curve did not reach to the death phase. Then, the

neutral and polar lipids in dried biomass were determined by gravimetric method after the

biomass was dried in Freeze dryer for 3 days as shown in Figure 6.4. With respect to the

presence of pH control when biomass was harvested at stationary phase, the biomass (higher

than 32%) with pH control contained higher total lipids than that (lower than 18%) without pH

91

control. With respect to DIC concentration, the total lipids were increased up to 18.2% (0.03 M

of DIC) from 10.4 and 10.8% (0 and 0.003 M of DIC) under without pH control, and the total

lipids was slightly increased up to 35.3% (0.03 M of DIC) to 32.0% (0.003 M of DIC) under

with pH control. Similarly, the neutral lipids increased up to 6.8% (0.03 M of DIC) from 3.7 and

2.3% (0 and 0.003 M) under without pH control, and neutral lipids increased up to 24.4% (0.03

M of DIC) form 4.6 % (0.003 M of DIC) with pH control. In addition, most of both total and

neutral lipids were reduced when the biomass was harvested at death phase compared to be

harvested at stationary phase because the metabolism of lipid accumulation might be inhibited at

death phase. In fact, the neutral lipids in S4 culture a slightly increased rather than that in S5

culture because the biomass in S4 and S5 was not reached to death phase until the final harvest

time. Therefore, it was noted that a high DIC might promote neutral lipid accumulation in N.

oleoabundans and pH control must be performed to maximize both total and neutral lipids.

Additionally, the biomass must be harvested at stationary phase but not death phase.

6.4. Conclusions

For the first-stage phase, a high DIC under pH control can significantly increase the

growth rate of green microalga N. oleoabundans in open systems. For the second-stage phase, a

high DIC under pH control can also significantly increase both total and neutral lipids

accumulation in microalgal cell. In addition, the loss of lipids in microalgae cell can prevent by

harvesting biomass at stationary phase. Consequently, the optimal growth and lipid

accumulation conditions for two-stage phase process can be determined in this study with respect

to DIC concentration, pH and harvest time.

92


This study was supported by the Metropolitan Sewer District of Greater Cincinnati

(MSDGC) under Master Services Agreement No. 85X10431, Task Order No. 0210000209 and

the NSF REU Site in Sustainable Urban Environments, Grant ID No. EEC-0851986. The

authors appreciate their financial support.

6.6 References

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Grove, Ill, 2002.

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96

Summary and Suggested Future Studies4 Chapter 7

7.1. Summary

The primary objectives of this study were to investigate the autotrophic growth and lipid

accumulation in freshwater microalgae under different cultivation conditions, particularly in

terms of the availability of nutrients (i.e. dissolved inorganic carbon (DIC), nitrogen, and

phosphorus), a degree of mixing, pH and light intensity. Below is a summary of the studies

conducted during my Ph.D. study.

1. Feasibility of using C. vulgaris to remove nitrogen and phosphorus in the secondary

wastewater effluent collected from a local wastewater plant

1.1 One of nutrients between nitrogen (NH3/NH4+) and phosphorus (PO4

3-) present in the

secondary municipal wastewater could be a limiting substrate for the growth of C. vulgaris

depending on their initial concentrations.

1.2. The nutrient removal rate could be increased by using a high initial algal cell density and

CO2(g) supply to control the pH at ~7. Almost all residual nitrogen and phosphorus present in

the secondary wastewater effluent could be removed within 48 hrs.

1.3. The Monod equation was used to express the growth kinetics for C. vulgaris depending

on a limiting substrate.

2. Study of the growth kinetics for C. vulgaris in terms of DIC concentrations under no

mixing condition

4 4 Part (Abstract and Conclusion) of the content in this chapter has been published in Kim, J.; Liu, Z.; Lee, J.-Y.; Lu,

T., Removal of nitrogen and phosphorus from municipal wastewater effluent using Chlorella vulgaris and its growth

kinetics. Desalination and Water Treatment 2013, 1-7; Kim, J.; Lee, J.-Y.; Ahting, C.; Johnstone, R.; Lu, T., Growth

of Chlorella vulgaris using sodium bicarbonate under no mixing condition. Asia-Pacific Journal of Chemical

Engineering 2014, n/a-n/a.; Kim, J.; Lee, J.-Y.; Lu, T., Effects of dissolved inorganic carbon and mixing on

autotrophic growth of Chlorella vulgaris. Biochemical Engineering Journal 2014, 82 (0), 34-40.

97

2.1 Sodium bicarbonate (NaHCO3) was found to be an excellent buffer that can help keep the

DIC concentrations in the culture medium high for open pond systems and the pH of the medium

appropriate for the growth of C. vulgaris.

2.2 The growth of C. vulgaris under no mixing condition was not significantly influenced

with respect to different DIC concentrations. The external mass transfer of DIC from the culture

medium to the algal cell surface was not a rate-limiting step because the estimated DIC

concentrations at the algal cell surface were close to that of the culture medium.

3. Study of the growth kinetics for C. vulgaris in terms of DIC concentrations under mixing

conditions

3.1 High DIC concentrations retained using NaHCO3 under mixing significantly enhanced

the growth kinetics for C. vulgaris because suspended algal cells could actively consume DIC

concentration for photosynthesis.

3.2 A mass-transfer study confirmed that the external mass-transfer resistance for DIC under

mixing was not a rate-limiting step for the growth of C. vulgaris with the use of NaHCO3 in the

medium. Therefore, it was found that the intracellular mass transfer and/or photosynthesis inside

the algal cell must be a rate-limiting step under no mixing condition.

3.3 The use of NaHCO3 along with CO2 gas under agitation could significantly enhance the

growth rate by overcoming the DIC limitation for photosynthesis. However, the growth was

eventually limited by light availability due to a shading effect derived from the high cell density.

3.4 The results also suggest that the carbon concentrating mechanism for microalgal growth

can be overridden under high DIC influx in an engineering system using CO2 gas supply in

conjunction with NaHCO3.

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3.5 A high concentration of NaHCO3 helps increase a DIC concentration for photosynthesis,

but was found to be limited by its salinity generated by Na+ for this freshwater green alga, C.

vulgaris.

4. Study of the effects of light intensity on the growth of C. vulgaris under nutrient-

sufficient conditions

4.1 The growth kinetics for C. vulgaris was dependent on algal cell density (photolimitation)

and high light intensity (photoinhibition). These two effects should be taken into account to

construct a growth model for cultivation.

4.2 The model developed could predict the growth of C. vulgaris in terms of incident light

intensities and reactor sizes with a reasonably good accuracy.

4.3 The model could be used to predict the growth of C. vulgaris for different reactor types

by incorporating hydrodynamics into the model.

5. Study of the effects of DIC, pH and harvest time on lipid accumulation in N.

oleoabundans under nitrogen deprivation.

5.1 High DIC concentrations with mixing significantly enhanced the growth rate of N.

oleoabundans under a nutrient-sufficient condition similar to the growth of C. vulgaris.

5.2 Total lipid accumulation in N. oleoabundans under nitrogen deprivation was significantly

increased with pH control for the culture medium.

5.3 Neutral lipid accumulation in N. oleoabundans under nitrogen deprivation was

significantly enhanced by increasing the DIC concentration in the culture medium.

99

5.4 The biomass should be harvested at the stationary phase for maximum lipid accumulation

because total and neutral lipid contents start to decrease at the death phase.

7.2. Suggested future studies

1. In this study, the external mass-transfer resistance for DIC was found to be negligible,

suggesting that intracellular DIC mass transfer and/or photosynthesis reaction should be a rate-

limiting step for the autotrophic growth. However, intracellular DIC mass transfer with respect

to different DIC concentrations was not previously reported. The intracellular DIC mass transfer

at a molecular level and its subsequent impacts on photosynthesis need to be studied for the

autotrophic growth.

2. In this study, it has also been found that pH and DIC concentration play a critical role in

the total and neutral lipid accumulations in N. oleoabundans under nitrogen deprivation.

However, little is known even about total lipid accumulation under nitrogen deprivation. It is

critical to study the metabolic pathways of total lipid accumulation and lipid speciation with

respect to pH and DIC concentration.

3. In this study, a semi-empirical growth model accounting for the autotrophic growth

coupled with photolimitation and photoinhibition was developed. The empirical lumped

parameter values determined for the model result from our incomplete understanding of

photolimitation and photoinhibition. A further study needs to be done for not only assessing the

applicability of the model but also an in-depth study of the light effects that can eventually lead

to a better descriptive mathematical model.

100

Bibliography of Jinsoo Kim EDUCATION

Chungnam National University (CNU), Daejeon, South Korea, BChE (Mar. 1996-Feb. 2003)

Seoul National University (SNU), Seoul, South Korea, MS (Mar. 2003-Feb. 2006)

University of Cincinnati (UC), Cincinnati, OH, US, Ph.D. (Sept. 2008-Apr., 2014)

PUBLICATIONS

SCI JOURNALS

[1] J. Kim, J.-Y. Lee, T. Lu, Effects of Dissolved Inorganic Carbon and Mixing on Autotrophic

Growth of Chlorella vulgaris, Biochemical Engineering Journal 82 (2014) 34-40

[2] J. Kim, J.-Y. Lee, C. Ahting, R. Johnston, T. Lu, Growth of Chlorella Vulgaris Using

Sodium Bicarbonate under No Mixing Condition, Asia-Pacific Journal of Chemical Engineering

(2014), In press.

[3] J. Kim, Z. Liu, J.-Y. Lee, T. Lu, Removal of Nitrogen and Phosphorus from Municipal

Wastewater Effluent Using Chlorella vulgaris and Its Growth Kinetics, Desalination Water

Treatment (2013), 1-7.

[4] X. Li, Z. Liu, J. Kim, J.-Y. Lee, Heterogeneous Catalytic Reaction of Elemental Mercury

Vapor over Cupric Chloride for Mercury Emissions Control, Applied Catalysis B:

Environmental (2013) 401-407.

[5] J. Kim, J. Yi, M.D. Ward, W.S. Kim, Separation of Dimethylcyclohexane Stereoisomers by

Selective Guest Inclusion of Host Compound of Guanidinium o-Terphenyl-4, 4'-Disulfonate,

Separation and Purification Technology 66 (2009) 57-64.

[6] J. Kim, J. Yi, W.S. Kim, Thermal Method for Prediction of Host Selectivity to Guest

Isomers, Journal of Chemical Engineering of Japan 42 (2009) 728-732.

[7] J. Kim, S.O. Lee, J. Yi, W.S. Kim, M.D. Ward, Mechanistic Study on Selective Inclusion of

Xylenes into Guanidinium p-Toluenesulfonate Host Frame, Separation and Purification

Technology 62 (2008) 517-522.

[8] I.H. Choi, Y.H. Kim, C.M. Kim, J. Kim, K.H. Choi, J.H. Yi, Site-Defined Micropatterning

Using Atomic Force Microscopic Lithography, Key Engineering Materials 277 (2004) 903-906.

NON-SCI JOURNALS

[1] J. Kim, B.P. Lingaraju, R. Rheaume, J.Y. Lee, K.F. Siddiqui, Removal of Ammonia from

Wastewater Effluent by Chlorella vulgaris, Tsinghua Science and Technology 15 (2010) 391-

396.

PROCEEDINGS

[1] Kim, J.; Lee, J.-Y.; Lu, T. Use of Sodium Bicarbonate for Efficient Carbon and Water

Management for Autotrophic Microalgae Cultivation in Open Pond System, Paper # 13336,

A&WMA’s 106th

Annual Conference, Chicago, IL, June 25-28, 2013.

[2] Kim, J.; Lee, J.-Y.; Lu, T. Effects of Dissolved Inorganic Carbon and Mixing on Growth and

Lipid Formation of Chlorella Vulgaris, Paper # 13330, A&WMA’s 106th

Annual Conference,

Chicago, IL, June 25-28, 2013.

[3] Lu, T.; Kim, J.; Lee, J.-Y.; Dunlap, P.; Shaw, A.; Barnard, J.; George, B.; Parrott, J.; Metz,

D., Investigation of the Synergistic Relationship between Nutrient Removal and Algae Growth in

Municipal Wastewater Treatment Plants, WEF/IWA Nutrient Removal and Recovery 2013

Conference, Vancouver, July 28-31, 2013.

101

[4] K.F. Siddiqui, J. Kim, B.P. Lingaraju, J.-Y. Lee, Removal of Nitrogen (NH3+/NH4) from

Wastewater by Chlorella vulgaris, Proceedings of the Water Environment Federation (2011)

178-184.

[5] J. Kim, J.-Y. Lee, K.F. Siddiqui, Effects of Total Inorganic Carbon on Growth of Chlorella

vulgaris, American Institute of Chemical Engineers, 2010, pp. a126/121-a126/126

[6] J. Kim, J.-Y. Lee, Growth kinetic study of Chlorella vulgaris, American Institute of

Chemical Engineers, 2009, pp. kim1/1-kim1/6.

KOREAN JOURNALS

[1] B. Kwak, J. Kim, J. Joo, J. Lee, J. Kim, Y. Kim, and J. Yi, Simulation of Plume Length

Induced by Orimulsion Combustioon, Clean Technology 14 (2008) 136-143.

[2] J. Kim, J. Park, J. Yi, and W. Kim, Molecular Separation of Dibromobenzene Isomers by

using Selective Guest Inclusion of G2NDS Host Framework, Korean Chemical Engineering

Research 45 (2007) 487-492.

[3] Y. Kim, J. Ki, J. Joo, J. Lee, J. Kim, B. Kwak, J. Jeong, S. Park, J. Yi, Investigation

of :Plume Opacity Induced by the Combustion of Orimulsion, Journal of Korean Society of

Environmental Engineers, 29 (2007) 297-303.

[4] S. Lee, J. Kim, C. Yun, and J. Yi, Adsorption Characteristics of the Sericite and Diatomite

for Ammonia Gas, Clean Technology 12 (2006) 175-181.

PATENTS

[1] U.S. Kim, J.H. Lee, S.O. Lee, J. Kim, M.D. Ward, J.H. Hwang, Selective Separation Method

for Xylene Isomers using Guanidinium p-Toluene Monosulfonate, Kyunghee University,

Industry-Academy Cooperation Foundation, S. Korea . 2009.

[2] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method For Selectively

Separating Xylene Isomers using Guanidinium m-Terphenyl-4,4'-Disulfonate, Kyunghee

University, Industry-Academy Cooperation Foundation, S. Korea . 2009.

[3] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method For Selectively

Separating Xylene Isomers by Selective Inclusion With Host Molecule of Guanidinium 4-

Chlorobenzenesulfonate, Kyunghee University, Industry-Academy Cooperation Foundation, S.

Korea . 2009.

[4] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method for Selectively

Separating Xylene Isomers by using Guanidinium 4-Iodobenzenesulfonate, Kyunghee University,

Industry-Academy Cooperation Foundation, S. Korea . 2009.

[5] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method for Selectively

Separating Xylene Isomers by Selective Inclusion Of Guanidinium 4-Bromobenzene Sulfonate,

Kyunghee University, Industry-Academy Cooperation Foundation, S. Korea . 2009.

[6] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method for Separation of

Xylene Isomer by Selective Inclusion of Guanidinium Biphenyl-2,2'-Diyl Dimethanesulfonate,


[7] U.S. Kim, J.H. Lee, J. Kim, S.O. Lee, M.D. Ward, J.H. Hwang, Method for Separation of

Xylene Isomers by Selective Inclusion of Guanidinium o-Terphenyl-4,4'-Disulfonate, Kyunghee

University, Industry-Academy Cooperation Foundation, S. Korea . 2009.

[8] U.S. Kim, J. Kim, J.H. Lee, S.O. Lee, S.G. Kim, M.D. Ward, Method For Separating e,e-

Dimethylcyclohexane Stereoisomer rrom Dimethylcyclohexane Mixture by using Guanidinium

102

o-Terphenyl 4,4'-Disulfonate Host, Kyunghee University, Industry-Academy Cooperation

Foundation, S. Korea . 2009.

[9] U.S. Kim, J. Kim, J.H. Lee, S.O. Lee, S.G. Kim, M.D. Ward, Method for Separating p-

Xylene Stereoisomer from Xylene Mixture by using Guanidinium 4-Chlorophenethyl

Monosulfonate Host, Kyunghee University, Industry-Academy Cooperation Foundation, S.

Korea . 2009.

[10] U.S. Kim, J. Kim, J.H. Lee, S.O. Lee, S.G. Kim, M.D. Ward, Method For Separating p-

Xylene From Xylene Mixture using Guanidinium 2-(2-Chlorophenyl) Ethylsulfonate Host,


[11] U.S. Kim, J. Kim, J.H. Hwang, M.D. Ward, K.T. Holman, Method For Selectively

Separating Xylene and Ethyl Benzene from Mixture, Kyunghee University, Industry-Academy

Cooperation Foundation, S. Korea . 2009.

[12] U.S. Kim, J. Kim, J.H. Hwang, M.D. Ward, K.T. Holman, Method for Selectively

Separating Xylene and Ethyl Benzene trom the Mixture , Kyunghee University, Industry-

Academy Cooperation Foundation, S. Korea. 2009.

Awards

[1] URC summer fellowship from the University of Cincinnati, 2010.

effects of dissolved inorganic carbon, ph, and light on growth and lipid accumulation in

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