Microalgae Growth

on Crude Glycerol
By: Kalvin Weeks
What are
microalgae?
Microalgae are unicellular
organisms, usually autotrophic,
capable of growing in the most
diverse environments found
across the earth
- Responsible for petroleum deposits
- Vast number of species estimated to be in the millions
- Characteristics comparable to autotrophs and heterotrophs

Microalgal Advantages
Can be grown on un-arable land, grown in salt
water bodies (oceans and bays), and grown on
water unfit for human consumption or agriculture

These organisms can reach high densities per unit
area and produce many marketable products such
as:
o Biomass (protein feedstock for animals and humans)
o Lipids (used as a feedstock for production of biofuels)
o Health Additives (omega-3 fatty acids)


Crude Glycerol
Crude Glycerol or bottoms is a
co-product of biodiesel
production

Formed when methanol
replaces glycerol, the 3 chain
carbon backbone of
triglycerides (oils).

o High energy density
o Low cost
o Contains impurities (methanol and soaps)
Biodiesel
Biodiesel
Process
Triglyceride
Free Fatty Acid
Glycerol
Hypothesis

Algae can be grown on crude glycerol to take
advantage of this low-cost, energy dense carbon
source.

Mixotrophic growth using crude glycerol will result in
higher growth rates and cell densities.


Objectives


Evaluate crude glycerol as a source of
carbon for algal growth which will result in:

o Enhanced algal growth

o Enhanced lipid production

o Economical source of energy dense carbon

Methodologies

Algae were grown under laboratory conditions

o Autotrophically and mixotrophically grown with 12 hours light/ 12
hours dark

o Heterotrophically grown in black flasks to prevent light penetration

o Temperature kept constant in both containers at 30C

o Aerated and shaken daily

Variables
Carbon sources:
- Crude Glycerol
- Pure Glycerol
Concentrations:
- .1 g/L (mixotrophic)
- 1 g/L (heterotrophic)

Controls were an autotrophic triplicate
with no additives, and a heterotrophic
triplicate with no additives


1
st
Triplicate 2
nd
Triplicate 3
rd
Triplicate 4
th
Triplicate 5
th
Triplicate 6
th
Triplicate
Autotrophic
Control
Mixotrophic
Pure Gly.
Mixotrophic
Crude Gly.
Heterotrophic
Control
Heterotrophic
Pure Gly.
Heterotrophic
Crude Gly.
200mL Algae 200mL Algae

200mL Algae

300mL Algae

300mL Algae

300mL Algae

No additions
.1 g/L Pure
Glycerol
.1 g/L Crude
Glycerol
No additions 1 g/L Pure
Glycerol
1 g/L Waste
Glycerol

o Optical Density
Measured at 545nm and 680nm

o pH

o Lipid stain analysis
Used Nile Red stain dissolved in acetone
Fluoresces yellow under UV illumination

Measurements
0.2
0.4
0.6
0.8
1
1.2
1.4
1 2 3 4 5 6 7 8 9 10
O
p
t
i
c
a
l

D
e
n
s
i
t
y

@

5
4
5
n
m


Time (days)
Mixotrophic Growth
Control Pure Glycerol Crude Glycerol
Error Bars 1 STD
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1 2 3 4 5 6 7 8 9 10
O
p
t
i
c
a
l

D
e
n
s
i
t
y

@

6
8
0
n
m

Time (days)
Mixotrophic Growth
Control Pure Glycerol Crude Glycerol
Error Bars 1 STD
- Crude Glycerol grew to higher densities than both the control and pure
glycerol

- Fascinating find since impurities show to result in increased growth
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
1 2 3 4 5 6 7 8 9 10
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g
/
L
)

Time (days)
Mixotrophic Concentration (g/L) from OD @ 545nm
Control Pure Glycerol Crude Glycerol
Error Bars 1 STD
1
1.2
1.4
1.6
1.8
2
2.2
2.4
2.6
2.8
1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
O
p
t
i
c
a
l

D
e
n
s
i
t
y

@

5
4
5
n
m

Time (days)
Heterotrophic Growth
Control Pure Glycerol Crude Glycerol
Error Bars 1 STD
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1 2 3 4 5 6
C
o
n
c
e
n
t
r
a
t
i
o
n

(
g
/
L
)

Time (days)
Heterotrophic Concentration (g/L) vs. Days from OD @ 545nm
Control Pure Glycerol Crude Glycerol
Error Bars 1 STD
In heterotrophic experiments, crude glycerol performed better at cell
growth rate and reached higher cell densities.
Lipid Stain
Mixotrophic Crude Gly. 4 days into experiment
Mixotrophic Crude Gly. 4 days into experiment
Mixotrophic Crude Gly. 4 days into
experiment
Heterotrophic Crude Gly. Final Day
Heterotrophic Crude Gly. Final Day
Heterotrophic Crude Gly. Final Day
Results
Crude Glycerol outperforms both control and pure
glycerol in both mixotrophic and heterotrophic trials

Growth Rate: (calculated by (day 2 day 1)+ (day 3
day 2).etc. and then divided by total days)

Autotrophic
Control
Mixotrophic
Pure Gly.
Mixotrophic
Crude Gly.
Heterotrophic
Control
Heterotrophic
Pure Gly.
Heterotrophic
Crude Gly.
.040 g/L/day .047 g/L/day .076 g/L/day -.003 g/L/day .023 g/L/day .095 g/L/day
1.9x the
growth rate
of control

4.1x the
growth rate
of pure gly.

Growth rates for crude glycerol is higher than all
other trials
o Initial Growth rates for heterotrophic trials were much higher than
average growth rates: 0.36 g/L/day

Studies of optical densities shows a higher cell
density present in flasks with crude glycerol

The use of crude glycerol promotes lipid
accumulation
o Excess carbon is used up and stored in oils


Conclusions

Algae can grow on crude glycerol
o Crude glycerol resulted in higher growth rates and
cell densities

Growth on crude glycerol resulted in
lipid synthesis

Heterotrophic growth conditions
show a more rapid growth rate

What does this mean?
Algae can first be grown to a maximum cell density with
natural sunlight and mediums (Landfill Permeate)

Collected into a mixotrophic system
o Crude glycerol is added
o Algae will grow on both sunlight and surplus crude glycerol

Mixotrophic system transformed to Heterotrophic
o Algae become stressed and immediately absorb crude glycerol and
store in the form of lipids

Results
- Increased cell density
- Increased volume of lipids per unit volume of algae
- Cultivation system is more optimized, more efficient, resulting in a
more economical production system



This algae is cheaper to process and contains more
marketable items per unit weight

Further steps will include processing the lipids into
biodiesel, which will result in more crude glycerol
which can be recycled back into the cultivation
system

Once a burden, crude glycerol is an economical
choice for algal production