For this Fall 2020 project, our team worked on growing heterotrophic and autotrophic algae in a batch system.

1. Kinetics Project
By: Courtney Kittel, Rachel Burger, & Caroline Packard

2. Introduction & Background
● All photosynthetic microbes, such as green algae, gain energy from solar
radiation/sunlight.
● Most photosynthetic microbes are also autotrophic, meaning they derive their
carbon from inorganic carbon sources. (2)
● For all autotrophs, the ATP and NADPH produced from the energy yielding
catabolic reactions are used in the anabolic reactions to reduce CO2 to yield
organic carbon compounds for production of new cells.(2)
● Many algal species are also able to grow as organoheterotrophs, meaning they
can grow without sunlight and they can derive their carbon from organic carbon
sources such as glucose.

3. The Reactors
● For our project, we wanted to make two algae batch reactors.
○ One reactor will have algae growth medium and stay in the sunlight, while the
other reactor will have glucose medium and be covered in tinfoil.
○ We wanted to see how well the algae grows depending on the energy and carbon
source.
● The algae reactor in the sunlight will be photoautotrophic because it will use light for its
energy source and CO2 for its carbon source (as mentioned before).
○ The reactor will also have a cap on it to simulate an anaerobic environment.
● The algae reactor with glucose and no sunlight will be chemoorganoheterotrophic
because it will use glucose for its energy and organic carbon source.
○ The reactor will stay open to simulate an aerobic environment.
● Both reactors will be given 9 days to grow after inoculation, with samples being taken
each day.

4. Measuring Algae Growth (XB)
● We measured optical density (OD) of both reactors everyday and used
calibration curves to calculate the TSS concentrations throughout the week.
● A TSS calibration curve from Lab 5 will be used for the algae in sunlight
● The TSS calibration curve for the algae with no sunlight had to be established
during our experiment
● The spectrophotometer was used to measure OD; it was set at 680 nm for the
algae in glucose and 750 nm for the algae in sun.

5. Measuring Substrate Utilization
● For the algae in glucose reactor,
○ We measured the COD concentration everyday. In order to do this, 0.5 mL of
sample and 2 mL of water were added to COD tubes. After digestion, the
absorbance values of the tubes were measured with a spectrophotometer set
at 600 nm.
○ Using a calibration curve from lab 6, the COD concentration could be
calculated with the measured absorbance values.
● For algae in sunlight reactor,
○ We measured the initial pH, and measured the alkalinity by titration with
0.02N H2SO4. The alkalinity was converted from mol equivalence/L to mg/L
CaCO3.
○ The pH was used to calculate the concentration of H+ and OH-. With the
concentration and alkalinity, the total inorganic carbon (Ct) can be calculated.
○ These measurements were done every other day, totaling 5 measurements

6. Results & Calculations
Algae in Sunlight
Days: 1 2 3 4 5 6 7 8 9
OD @
750nm
0.063 0.088 0.106 0.124 0.139 0.141 0.135 0.151 0.174
TSS
calc.
[g/L]
0.063 0.094 0.113 0.133 0.149 0.151 0.144 0.161 0.186
OD vs TSS calibration curve
for algae in sunlight

7. Results & Calculations for Algae in Sunlight
Days Initial
pH
Initial
pOH
Final
pH
Volume
titrated
[mL]
Initial
burette
readin
g [mL]
Final
burette
readin
g [mL]
1 10.36 3.64 4.48 5.0 33.5 38.5
3 10.36 3.64 4.50 5.6 32.7 38.3
5 10.76 3.24 4.45 6.2 30.0 36.2
7 10.89 3.11 4.45 6.1 38.5 44.6
9 11.01 2.99 4.52 5.8 32.5 38.3
Days H+
concentrati
on [mol/L]
OH-
concentr
ation
[mol/L]
Alkalinit
y [mol
eq./L]
Alkalinity
[mg/L]
Ct
[mol/L]
1 4.37E-11 2.29E-04 3.33E-03 1.67E+02 1.55E-03
3 4.37E-11 2.29E-04 3.73E-03 1.87E+02 1.75E-03
5 1.74E-11 5.75E-04 4.13E-03 2.07E+02 1.78E-03
7 1.29E-11 7.76E-04 4.07E-03 2.03E+02 1.65E-03
9 9.77E-12 1.03E-03 3.87E-03 1.93E+03 1.42E-03

8. Average Biomass Yield (YB) for Algae in Sunlight
YB average = 36.70 (gXB/L) / (g S/L)

9. Creating the TSS vs OD calibration curve
for the algae in glucose reactor
Sample ID Tin # Mass, dried
sample
+filter [g]
Mass,
clean filter
[g]
Volume
filtered [L]
Weight
gain/loss
[g]
Dry weight
g/L
Optical
density @
680nm
Blank 1 0 1.392 1.392 0.025 0 0.020 0
Blank 2 0-2 1.450 1.451 0.025 -0.001 -0.020 0
25% 25 1.438 1.432 0.025 +0.006 0.260 0.524
25% 25-2 1.431 1.426 0.025 +0.005 0.220 0.524
50% 50 1.128 1.123 0.015 +0.005 0.367 0.825
50% 50-2 1.404 1.392 0.025 +0.012 0.500 0.825
75% 75 1.433 1.423 0.015 +0.010 0.700 1.060
75% 75-2 1.416 1.414 0.020 +0.002 0.125 * 1.060

10. Results & Calculations
for Algae in Glucose
Days 1 2 3 4 5 6 7 8 9
OD @
680nm
0.057 0.120 0.248 0.664 0.940 1.090 1.160 1.270 1.300
TSS
calc.
[g/L]
0.033 0.070 0.144 0.385 0.545 0.632 0.673 0.736 0.754
OD vs TSS calibration curve for
algae in glucose reactor

11. Results & Calculations for Algae in Glucose
Days Absorbance
@600nm
COD
Concentration
[g/L]
Glucose
Concentration
[g/L]
1 0.179 2.964 2.777
2 0.259 4.288 4.019
3 0.055 0.911 0.853
4 0.127 2.103 1.971
5 0.049 0.811 0.760
6 0.059 0.977 0.915
7 0.046 0.762 0.714
8 0.028 0.464 0.434
9 0.012 0.199 0.186
COD vs Absorbance
calibration curve

12. Average Biomass Yield (YB) for algae in glucose
YB average=0.183 (g XB/L) / (g S/L)

13. TSS Comparison

14. Substrate (carbon) Comparison

15. Day 1: Algae on left, Glucose on right. Day 3: Algae on left,
Glucose on right.

16. Day 4: Algae on left, Glucose on right Day 5: Algae on left,
Glucose on right

17. Day 8: Algae on left, Glucose on right Day 9: Algae on left,
Glucose on right

18. STELLA Model for Algae in Sunlight
MBE wrt XB
+mu*XB-b*XB=dXB/dt
MBE wrt S
(-1/YB)*mu*XB=dS/dt

19. STELLA Model for Algae in Sunlight

20. STELLA Model for Algae in Glucose
MBE wrt XB
+mu*XB-b*XB=dXB/dt
MBE wrt S
(-1/YB)*mu*XB=dS/dt

21. STELLA Model for Algae in Glucose

22. Discussion & Conclusions
● Overall both reactors had algae growth, which was a success!
● The algae without sunlight grew at a faster rate and had higher TSS values than the algae in
sunlight.
● The algae in the sunlight was only able to grow during certain times of the day. Its sole
energy source was the sun, which is only available during the day. The algae with no sun and
with glucose had energy available to it all day, so it was able to grow at a faster rate.
● The substrate calculations also backs this up.
● The STELLA models don’t match up with the data we collected. The STELLA models are very
idealistic instead of realistic. It shows complete substrate utilization which almost never
happens.
● The biomass yields for our data and the STELLA models were also very different. For the
algae in sunlight, it had a higher YB than the STELLA model. For the algae without sunlight, it
had a lower YB than the STELLA model.

23. Citations
(1) Jesús Zambrano, Ivo Krustok, Emma Nehrenheim, Bengt Carlsson, A simple model for
algae-bacteria interaction in photo-bioreactors, Algal Research, Volume 19, 2016,
Pages 155-161, ISSN 2211-9264, https://doi.org/10.1016/j.algal.2016.07.022.
(http://www.sciencedirect.com/science/article/pii/S2211926416302570 )
(1) Drapcho, C. 2020. Biochemical Pathways-Lithotrophic and phototrophic growth. Unpublished
Lecture 10 Notes, BE 4100, Clemson University, Clemson SC.
(1) Drapcho, C. 2020. Modeling microbial growth with Monod model. Unpublished Lecture 12
Notes, BE 4100, Clemson University, Clemson SC.