Microalgae - A Potential Fresh Water

Microbial Agent For CO2 Sequestration

And Lipid Production

Dr. Raj Mohan B.

A s s o c i ate Pro fe s s o r,
Dep ar t m en t o f C he m i c a l E ng in e er ing
N IT K – S u rath k al , M anga lo re 5 7 5 0 2 5 ,
K a r n a t a k a, In d i a
Telephone: +91 824 2473605 Cellular Phone: +91 9739939986, Email: rajmohanbala@gmail.com
 Global Warming

“Burning Issue is not only bout Global Warming, it is also about what we are Burning”
 Assessment of Global Warming

Intergovernmental Panel on Climate Change (IPCC)

Assessment
First Report - 1990
Air temp 0.3 to 0.6 oC over the last 100 years, Sea level rise 6cm, 0.3 oC per decade , 20 cm by 2030, and
65 cm by the end of the next century

Second Report - 1995

CO2 is the most important contributor to anthropogenic forcing of climate change

Third Report - 2001

GHG and aerosols due to human activities, 1.4 to 5.8 oC over the period 1990 to 2100, and the sea level is
projected to rise by 0.1 to 0.9 metres over the same period

Fourth Report - 2007

Average global temperature increase 1906-2005 – 0.74 oC Expected Temperature rise up to the Year 2100
2.4 to 6.4 oC, Expected Sea Level rise 18 to 59 cm
National Renewable Energy Laboratory, USA
 Melting of ice glaciers in Himalayas (33 000
km2 )

Calculations indicated that 915 km2 of Himalayan

glaciers (test region) Spiti/Lahaul (H P, India) thinned
by an annual average of 0.85 m between 1999 and
2004.
Total loss of 3.9 km3 of water in 5 years

Although retreating glaciers would provide more

meltwater in the short term, the loss
of glacier "insurance" could become problematic over
the long term
 Hindu Kush-Himalayan region
Covers 8 countries across Asia
Major river systems -- The Ganges, Mekong, Yangtze, and Yellow
rivers
Sources of drinking water and irrigation supplies for roughly 1.5
billion people.   

Shigri Glacier in the Indian Himalaya: Mark

W. Williams, University of Colorado, Boulder

Water stored as glacial ice serve as the Himalayan region's

Glaciers in the eastern and central regions
of the Himalayas are retreating at rates that
have accelerated over the past century.

The glaciers of the Hindu Kush Himalayas

will dwindle in size until normal glacier
melt and can no longer contribute to the
region’s water supply.

The position of the end (terminus) of

Gangotri Glacier, India, between 1780 and
2001.

Image Source: U.S. Geological Survey

 Rising sea levels, stronger monsoons, and
deforestation

Ocean could rise in the next

100 years to 1 m higher
than the current sea level
Aslak Grinsted
Geophysicist
Centre for Ice and Climate
Niels Bohr Institute
University of Copenhagen, Denmark

Between 1993 and 2003, the global mean sea level rise of 3 mm/y. Losses from
glacial masses accounts for the mass of sea water increase and are responsible for 80
% of the average rise in sea level in recent years
 is i s
Cr
rgy
E ne
Growing World Energy Demand

Source: National Energy Technology Laboratory

 Coping with Global Warming

Greenhouse Gases Earth Interactions

Air interaction
S P
C R
CAUSE EFFECT
I E
E D
N I
Global
T Mitigation Strategy
C
Warming Climate Change Impacts -Adaptation
I Renewable sources
T
Crop Productivity
F I
Energy Efficiency Frequent Disasters
I V
Clean Coal Technology Water Scarcity
C E
Carbon Capture and Storage Vector borne diseases
(CCS)
 Green House Gas – CO2

The Energy generating plants contributes mostly to increasing atmospheric CO 2

concentrations.

CO2 concentration increased from 280 ppm to 390 ppm in the present.
Statistical Review
 Major and Minor Greenhouse Gases and
Global Warming Potential
Major greenhouse gas
concentrations of CO2, NOx,
CFCs, Methane have
increased 20-30% since pre-
industrial era

Derived from the Bern

carbon cycle model

The amount of heat trapped by a certain mass of a gas to that of the amount of heat trapped by a similar mass of
 CO2 Existence
CO2 remains in the atmosphere longer
than other major heat-trapping gases

10yrs for methane (CH4) (converts into

CO2) 100 yrs for nitrous oxide (N2O).

CO2: much of today’s emissions will

be gone in a century, but about 20 %
will still exist in the atmosphere
approximately 800 years from now
Water vapor is not consider as emission due to human
activities, and more or less its cycle is in few days
Climate Influence Between 1750 - 2055
 CO2 sequestration- Definition

Storage of carbon dioxide (usually captured

from the atmosphere) in a solid material
through biological or physical processes
 CO2 Sequestration: What Is It?
 Also known as “carbon capture”

 A geo-engineering technique for the long-term storage of carbon

dioxide (or other forms of carbon) for the mitigation of global warming

 More than 33 billion tons of carbon emissions (annual worldwide)

Ways that carbon can be stored (sequestered):

 In plants and soil “terrestrial sequestration” (“carbon sinks”)
 Underground “geological sequestration”
 Deep in ocean “ocean sequestration”
 As a solid material (still in development)
CO2 sequestration pathways
Terrestrial Carbon Sequestration
 The process through which CO from the
2
atmosphere is absorbed naturally through
photosynthesis & stored as carbon in
biomass & soils.

 Tropical deforestation is responsible for

20% of world’s annual CO2 emissions,
though offset by uptake of atmospheric
CO2 by forests and agriculture

 Ways to reduce greenhouse gases:

 Avoiding emissions by maintaining
existing carbon storage in trees and
soils
 Increasing carbon storage by tree
planting or conversion from
conventional to conservation tillage
practices on agricultural lands
Geological Sequestration
Midwest Geological Sequestration Consortium
(Illinois Basin)  

Injection of supercritical carbon dioxide began in

November 2011 at a rate of 1,000 metric tons per
day.

The project goal is to inject 1 million metric tons by

the end of 2014. Injection is at a depth of 7,000
feet and a full environmental monitoring program
in the subsurface and at the surface

Assess geological carbon sequestration options in the

60,000 square mile Illinois Basin (Within the
Basin are deep, noneconomic coal resources,
numerous mature oil fields and deep saline rock
formations with potential to store CO2)

Feb 2009: Successfully completed 8,000 ft deep

injection well. By 2013, a total of one million
metric tons of carbon dioxide.
Ocean Sequestration
 “Carbon is naturally stored in the ocean via two
pumps, solubility and biological, and there are
analogous man-made methods, direct injection and
ocean fertilization, respectively.

 Eventually equilibrium between the ocean and the

atmosphere will be reached with or without human
intervention and 80% of the carbon will remain in the
ocean. The same equilibrium will be reached whether
the carbon is injected into the atmosphere or the
ocean.

 The rational behind ocean sequestration is simply to

speed up the natural process.” Carbon sequestration
by direct injection into the deep ocean involves the
capture, separation, transport, and injection of CO2
from land or tankers
 
 1/3 of CO2 emitted a year already enters the ocean

 Ocean has 50 times more carbon than the

atmosphere

 "At great depths, CO2 is denser than sea water, and it

may be possible to store it on the bottom as liquid or
deposits of icy hydrates
 Carbon Capture Options
Post-Combustion
Clean gas CO2

Flue gas

ABS H STRP
X

Recirculating
(amine) solvent

Source: http://www.co2crc.com.au/
 Biofuels
Wide range of fuels which are derived from biomass

solid
biomass

liquid
fuels

various
biogases
 Generations of Biofuels

1st 2nd 3rd

From non-food crops,

From sugar, starch, stalks of wheat, corn, wood
vegetable oil or animal etc. Ex., bio hydrogen, bio From whole algae or
methanol, Bio DME,
fat. Ex., bio alcohols, Fischer-Tropsch diesel, bio
extracts of algae (also
green diesel, bio ethers, hydrogen diesel, mixed called oilgae)
biogas, syn gas alcohols and wood diesel,
cellulosic ethanol
Why Micro Algae?

 High Growth rate

 Minimal resource requirement
 High Photosynthetic efficiency
 Up to 70% of algae biomass is
usable oils
 Does not compete for land and
space with other agricultural
crops
 Can survive in water of high salt
content
 Why Micro Algae?
Benemann et al., 1997
 Continuous production biomass
 Ability to provide optimal nutrient levels (e.g., CO2, N, P, etc) at all times
 Absence of non-photosynthetic supporting structures
 Ability to adjust harvest rates
 Control over cell composition
Tapie & Bernard, 1988
 Carbon Content: 45–50% of the dry algal mass
 Theoretical CO2 requirement per g of algal biomass: 1.65–1.83 g
 Micro Algae as Biofuel
Crop Oil Yield (Gallons/acre)
Corn 18
Cotton 35
Soybean 48
Mustard seed 61
Sunflower 102
Rapeseed/ Canola 127
Jatropha 202
Oil palm 635
Algae (10 g/m2/day at 15% TAG) 1200
 Overview of micro-algae technology for
carbon sequestration

Algal Biotechnology Converts Flue Gases & Sunlight into Biofuels “Used” Algae have
through Photosynthesis Multiple Potential Uses

Cleaned
Gases
Sunlight Co-Firing
Power Plant / Green Power
Energy Source
Photo bioreactor
Esterification
Biodiesel

Flue
Gases Fermentation Ethanol

Drying Protein Meal

NOx + CO2 from
combustion flue Algal
gas emissions Biotechnology
Micro Algae
 Species Known Max CO2 Conc References
Tolerance Test
Cyanidium caldarium 100% Seckbach et al., 1971

Scenedesmus sp. 80% Hanagata et al., 1992

Chlorococcum littorale 60% Kodama et al., 1993

Synechococcus elongates 60% Miyairi, 1997

Euglena gracilis 45% Nakano et al., 1996

Chlorella sp. 40% Hanagata et al., 1992

Eudorina sp. 20% Hanagata et al., 1992

Dunaliella tertiolecta 15% Nagase et al., 1998

Nannochloris sp. 15% Yoshihara et al., 1996

Chlamydomonas sp. 15% Miura et al., 1993

Tetraselmis sp. 14% Matsumoto et al., 1995

 CO2 assimilation
The assimilation of CO2 is catalyzed by ribulose-1, 5-bisphosphate
carboxylase /oxygenase (Rubisco), which is the most abundant protein on
earth, with the double function of acting as mono oxygenase when O2 is the
substrate and as carboxylase when CO2 is the substrate (Watson et al., 1997).

The carboxylase reaction of Rubisco is usually the rate limiting step in

photosynthesis (Monorey and Somanchi, 1999)

High concentrations of the internal Ci upto 50 mM (the inorganic carbon

species including CO2, carbonic acid, bicarbonate anion, and carbonate anion)
are required for Rubisco co-catalyzed CO2 assimilation

CO2 in aqueous extracellular environment is usually only in the range of 10-12

µM
CO2 assimilation ….
Algal Cultivation
 Open Pond & Photo Bioreactor
Parameter Open pond Closed photo bioreactor
Construction Simple More complicated-varies by
design
Cost Cheaper to construct, More expensive
operate construction, operation
Water losses High Low
Typical biomass concentration Low, 0.1-0.2 g/L High: 2-8 g/L
Temperature control Difficult Easily controlled
Species control Difficult Simple
Contamination High risk Low risk
Light utilization Poor Very high
C02 losses to atmosphere High Almost none
Typical Growth rate(g/m2-day) Low:10-25 Variable:1-500
Area requirement Large Small
Depth/diameter of water 0.3m 0.1m
Surface: volume ratio ~6 60-400
 Experimentation
1. Acclimatization of strains in 3% CO2 conditions

2. Study of growth response to varied CO2 concentrations (6%, 9%, 12%

and so on)

3. Selection of high CO2 resistant strains and sub culturing

4. Isolation of lipid producing microalgae by Nile Red Staining test

5. Development of Lab scale study on algal bioreactor

6. Culturing of isolated strains in bioreactor
7. Periodic Lipid extraction and its qualitative analysis by TLC and GC
 Generation of CO2 in a Two Tier Flask System

CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+ (1)

CO32- + H+ ↔ HCO3- (2)

In the two-tier flask system, the CO2 is utilized by the algae

continuously, thus favoring the backward reaction of equation
(1) resulting in production of more amount of CO2

Two tier flask system for photo-autotrophic growth containing

BG11 medium in upper compartment and KHCO3/ K2CO3
buffer mixture in the lower compartment.
 Static Flask and Two Tier Studies

Table: Establishment of Stock solution Buffer mixture CO2 partial

CO2 partial pressure by
KHCO3/ K2CO3 KHCO3/ K2CO3 pressure (%v/v)
KHCO3/ K2CO3 solutions
(molesL-1) (ml)

3.0 62/38 1.0 ± 0.1

3.0 73/27 2.0 ± 0.1
4.0 73/27 3.0 ± 0.1
 Physiological conditions
Media Used: Bolds Basal Media
Temperature: 220c
Light conditions: 450-500 lux
CO2 concentration: Ambient (0.03%)
Wavelength of OD: 680 nm
Incubation period: 15 days
The accurate CO2 partial pressure in headspace of the flask is optimized to 3% by using
buffer mixture and monitored by InfraRed CO2 sensors.

For 3% CO2, following buffer ratio is maintained. KHCO3/K2CO3 (4 molar concentration of

buffer) in the ratio 75ml + 25ml
 CO2 Fixation rate
Biomass productivity (P) was calculated using the equation,

P = Total biomass (g/L/day)

Total volume of media filtered X incubation period in days

Cb= 0.68 * OD680 (Molina Grima et al,. 1994b)

and

P= Cb (g/L/day)
incubation period in days
 
where,
Cb is the biomass concentration in g/L and OD680 is the absorbance at 680 nm.

CO2 fixation rate, PCO2 = 0.50 x P x 44 x 12-1 (g/L/day) (Ugwu et al., 2005)
 Comparison of Chlorophyll content for two tier flask system

Pigment-chlorophyll
3500

3000

2500

2000 Chlorophyll A
Chlorophyll content in Chlorophyll B
the biomass in mg/m3 1500 Total chlorophyll

1000

500

0
.03% 1% 2% 3%

%CO2
 Variation in production of various chlorophylls in E. gracilis in
the two tier flask system

2.5

2
Chlorophyll content (mg/l)

1.5

ChlA(mg/l)
ChlB(mg/l)
1
Total Chl(mg/l)

0.5

0
C 1% 2% 3%

% concentration of CO2
 Anti-oxidant activity for E. gracilis in two tier flask system

Antioxidant activity
0.4

0.35

0.3
FRAP in mM/g

0.25

0.2 FRAP(mM/g)

0.15

0.1

0.05

0
C 1% 2% 3%

% concentration of CO2
Reactor studies

A glass cylinder of 97cm length 8.2cm Øi and

9.9cm Øo with glass inlet at the bottom and one
outlet at the top

It was connected to two gas flow rotameters,

one for air and other for CO2.
 Effect of CO2 on Biomass Production

CO2 sequestration rate was

optimized by selecting suitable
flow rate, decrement in bubble
size, increasing resident time
and optimization of intensity
of light.

Initial experimental trials were

carried out to optimize the
concentration of CO2 for the
production of biomass data as
shown in Figure
The equilibrium used for this study and the equilibrium constants values are the same as
those used by Brune and Novak (1981) and Camacho Rubio et al. (1999).

CO2 + H2O ↔ H2CO3 ↔ HCO3- + H+

K1 = [HCO3- ] + [H+] = 10-6:381

[CO2]

HCO3- ↔ CO2 K2 ↔ [CO32- ] + [H+] = 10-10:377

[HCO3]

H2O ↔ [H+] + [OH-] Kw = [H+] [OH-] = 10-14

The inorganic carbon present in the microalgae culture can be expressed as:

[Ctotal] = [CO2] [HCO3- ] + [CO32-]

Taking into account the simplified alkalinity definition for the culture media:

[Alkalinity ] = [HCO3- ] + 2[CO32-] + [OH-] - [H+]

CTotal = [alk] - [OH-] + [H+]
(α1 + 2 α2 )

Combining Eqs. we can relate the total inorganic carbon in the culture media as a function of
the Alkalinity and pH.

Where:

α1 = [1 + K2 + [H+] ]
[H+] K1

α2 = [1 + [H+]2 + [H+] ]
K1 K2 K2

The evolution of the inorganic carbon dissolved in the culture media during the pH raise
observed in the pH profile can be associated to the CO 2 photosynthetic consumption and CO2
stripping, therefore the next expression can be written:

dCTotal = KLa [CO2*] – [CO2] + Rphoto

dt
where KLa (h-1) is the overall volumetric mass transfer coefficient for the CO 2, [CO2] (mol/l)

represents the saturation concentration of CO2 in the liquid phase and R photo (mol CO2/l h)

represents the CO2 consumption rate by the microalgae photosynthetic activity.

The [CO2] can be calculated using the CO2 solubility Henry’s constant in medium and the

partial pressure of CO2 in the air injected into the reactor

[CO2*] = PCO2
H

The evolution of the dissolved inorganic carbon in the culture media during the pH raise
observed in the pH profile is caused by the CO2 stripping, therefore:

dCTotal = KLa [CO2*] – [CO2]

dt

The use of the pH profiles during the light day and the K La value permit to obtain relevant
information about the behaviour of the microalgae/ photobioreactor system related to the CO 2
net balance.
 The evolution of η = CO2 uptaken x 100
CO2 uptaken + CO2 stripped in the bubble column per hour

Chlorophyll a = [11.85 x(OD664) -1.54 x (OD647) - 0.08 x (OD630)]x V1

(mg/m3) V2 x L

Chlorophyll b = [20.13 x (OD645) - 4.19 x (OD661.5)] x V1

(mg/m3) V2 X L

Total chlorophyll = [7.05 x (OD661.5) +18.09 x (OD645)] x V1

(mg/m3) V2 x L

Carotenoid = [1000 x (OD470) + 1.9CA – 63.14CB] x V1

(mg/m3) V2 x L

Where V1= volume of supernatant after centrifugation (ml)

V2= volume of culture filtered (L)
L= path length of cuvette used in spectrophotometer (cm)
CA= Chlorophyll A content (mg/m3)
CB = Chlorophyll B content (mg/m3)
Effect of Bubble Diameter on Biomass Effect of Flow Rate on Biomass Production
Production and CO2 Fixation rate and CO2 Fixation
 Effect of Light Intensity on Biomass
Production and CO2 Fixation rate

Light intensity plays an important role in photosynthesis, in plants

and microalgae.

Five trials were performed at optimized CO2 concentration of 35.9%

and at three different light intensities (1.72, 3.28, 5.0, 6.72 and 8.44
Klux)
 Gas hold up and Volumetric Flowrate

The superficial gas velocity

in the photobioreactor was
studied to analyze its effect
on the gas holdup and
mass transfer coefficient.
The experimental gas
holdups for three different
superficial gas velocities
 Volumetric Mass Transfer Coefficient

x 6 𝜀𝑔
𝑎= Hughmark
𝑑𝐵

√
𝑑𝐵 𝑉 𝑡 𝜌 𝐿 εg = Vg
𝑅𝑒𝐵 = 2 𝜎 𝑔 𝑑𝐵
𝜇𝐿 𝑉 𝑡= + 0.3 + 2Vg
𝑑𝐵 𝜌 𝐿 2 Carvalho & Malcata, 2001
 Effect of CO2 on Biomass Productivity

Five experimental trials were

performed to check the effect
of different concentrations of
CO2 (0.3%, 23.12%, 35.9%,
45.08% and 51.95%) on
pigments and antioxidant
activity at the optimized light
intensity of 5.18 Klux in the
sparger 2 with mesh at the
optimized flow rate of 1.5 lpm
 Effect of Light Intensity of Productivity
At optimized CO2 conc of 35.9%
the productivity of three
different light intensities viz.
1.72, 3.28, 5.0, 6.72 and 8.44
Klux were analyzed as shown in
figure

It was observed that at 5.0 Klux

the biomass production rate and
CO2 sequestration rate was high
than at the other trials.

Though the biomass production

rate and CO2 fixation rate at 6.9
Klux light intensity was the same
as that of 5.0 Klux, it had a
negative effect on the pigment
production
 Effect of CO2 on pigments and antioxidant activity
It was observed that the production of
chlorophylls was not influenced by CO2 .

Pigment production was successfully

increased with increasing growth of
Euglena gracilis.

Production of chlorophyll a and

chlorophyll b increased as the CO2
concentration increased upto the 3rd trial
then it decreased.

It clearly indicates the response of Euglena

gracilis against the approach of CO2
towards the tolerance limit (45% CO2 in
air).

The antioxidant activity increased in the

2nd and 3rd trial compared to the 1st. Along
with growth of Euglena gracilis for CO2
sequestration level of upto 35.9% beyond
which it decreased.
Growth Kinetics of Euglena gracilis in a photobioreactor
dX  X  X 0e max t
 max 1.0  X X (t ) 
The parameter μmax in Logistic equation dt X max   X 
 
1   0  1  e maxt 
was determined from the plot ln (X/(Xmax-  X max 
X)) vs time (t).
μmax was found to be 0.87 (day-1).

X0 and Xmax was found to be 0.038 (g/L),

1.665 (g/L) respectively from intercept.
Using the values of μmax and X0 biomass
concentration was predicted according to
equation.
The comparison of experimental and the
logistic model predicted biomass
concentration.
The correlation coefficient (R2) value of
0.992 was obtained with logistic model
for biomass.
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Thank You