NASA ICED Challenge Research ​ Your Last Name 1

Author(s) / Student(s): Your Full Name(s)

Teacher: Mr. Richard Wilczewski

Course: Chemistry CHE 115

Program: Dr. Camarda’s Innovative Conceptual Engineering Design / NASA Challenge

Rowan College at Burlington County, Mount Laurel, NJ

Date (Day, Month, Year)

Title

Abstract

Motivation

Research Questions

Literature Review

A study was done to evaluate the growth of Chlorella sp. on wastewaters. Four different parts of
the process of treating the sample of wastewaters were recorded, as well as how well the algae
removed nitrogen, phosphorus, chemical oxygen demand and metal ions from the wastewaters.
Results indicated that algae could prove valuable in removing nutrients in municipal wastewater,
as well as providing biomass feedstocks for renewable energy.

Research done by Amman and Lynch evaluated the amount of oxygen produced by algae when
multiple concentrations of CO2 were present. They found that 2% CO2 flowing at 320 mL per
minute, 3% CO2 at 135 mL per minute and 4% CO2 at 55 mL per minute yielded optimal CO2
concentrations.

Drapcho and Brune conducted an experiment to determine the productivity of O2 via solar
radiation in algae cultures. The introduction of CO2 caused a change in the production of O2.
With the original blue-green algae the O2 production rate was 0.096 mg O2/mg TSS per hour at

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20 degrees celsius, but with the introduction of CO2 to green algae at 0.6-1.2 mmol/l per day
increased the O2 production to 0.14mg O2/mg TSS per hour. The max growth composition
rates for both was 0.077 and 0.12/h for blue-green, and green algae.

Researchers at the Chinese Academy of Science compared a two-stage process to produce

hydrogen and methane from algae to a one stage process. The two stage process generated
46mL of hydrogen gas and 396mL of methane. The methane production was 22% higher than
the one stage process. The energy efficiency also went up by 14% in the two stage process
compared to the one. It was also found that in the two stage process if you used the same batch
multiple times the methane production rate improves.

Sialve et. al examined the anaerobic digestion of algae to solve waste issues, and the
economical and energetic balance of this process. They found that the process of anaerobic
digestion can produce more energy from algae than the cell lipids themselves. They also found
that best rate for anaerobic digestion was when the cell lipid content does not go over 40%.

Hansen and Viamajala isolated three strains of algae from wastewater lagoons in Utah on
anaerobic digester effluent, and tested their growth on anaerobic digester effluent under varying
conditions. They found that growth rates and biomass production increased after a period of
adaptation to the effluent, as well as with supplementation of light, with the best growing strain
producing 2.71 g/L of biomass on average.

Researchers at University of Florida investigated the feasibility of anaerobic digestion for

organic waste treatment on space missions. They found conversion efficiencies of 75% and
85%, with residence times of 2-3 weeks, for various blends of organic waste, without need for
pH control.

Breshonda:

Cultivation of Green Algae Chlorella sp. in Different Wastewaters from Municipal Wastewater
Treatment Plant

https://link.springer.com/article/10.1007/

A study was done to evaluate the growth of algae Chlorella sp. on wastewaters. Four different
parts of the process of treating the sample of wastewaters were recorded, as well as how well
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the algae removed nitrogen, phosphorus, chemical oxygen demand and metal ions from the
wastewaters. Results indicated that algae could prove valuable in removing nutrients in
municipal wastewater, as well as providing biomass feedstocks for renewable energy.

Jenna:

Bok Lee, Sun & Park, Jong. (1997). Carbon Dioxide Fixation by Algal Cultivation Using
Wastewater Nutrients. Journal of Chemical Technology and Biotechnology - J CHEM TECHNOL
BIOTECHNOL. 69. 451-455. 10.1002/(SICI)1097-4660(199708)69:43.3.CO;2-D.

https://www.researchgate.net/publication/255575010_Carbon_Dioxide_Fixation_by_Algal_Cultiv
ation_Using_Wastewater_Nutrients

Gas Exchange of Algae

III. Relation between the Concentration of Carbon Dioxide in the Nutrient Medium and the
Oxygen Production of Chlorella pyrenoidosa

http://aem.asm.org/content/15/3/487.short

A study was done by Amman and Lynch evaluating the amount of oxygen produced by algae
when multiple concentrations of CO2 were present. 2% CO2 flowing at 320 mL per minute, 3%
CO2 at 135 mL per minute and 4% CO2 at 55 mL per minute yielded optimal CO2
concentrations.

Andrew:
Sum-
Drapcho and Brune conducted an experiment to determine the productivity of O2 via solar
radiation in algae cultures. The introduction of CO2 caused a change in the production of O2.
With the original blue-green algae the O2 production rate was 0.096 mg O2/mg TSS per hour at
20 degrees celsius, but with the introduction of CO2 to green algae at 0.6-1.2 mmol/l per day
increased the O2 production to 0.14mg O2/mg TSS per hour. The max growth composition
rates for both was 0.077 and 0.12/h for blue-green, and green algae.

The partitioned aquaculture system: impact of design and environmental parameters on algal
productivity and photosynthetic oxygen production
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http://www.sciencedirect.com/science/article/pii/S014486099900028X

Eric:

Hydrogen and methane production from lipid-extracted microalgal biomass residues

Researchers at the Chinese Academy of Science compared a two-stage process to produce
hydrogen and methane from algae to a one stage process. The two stage process generated
46mL of hydrogen gas and 396mL of methane. The methane production was 22% higher than
the one stage process. The energy efficiency also went up by 14% in the two stage process
compared to the one. It was also found that in the two stage process if you used the same batch
multiple times the methane production rate improves.

http://www.sciencedirect.com/science/article/pii/S0360319910023608

Anaerobic digestion of microalgae as a necessary step to make microalgal biodiesel sustainable

Sialve et. al examined the anaerobic digestion of algae to solve waste issues, and the
economical and energetic balance of this process. They found that the process of anaerobic
digestion can produce more energy from algae than the cell lipids themselves. They also found
that best rate for anaerobic digestion was when the cell lipid content does not go over 40%.

http://www.sciencedirect.com/science/article/pii/S0734975009000457

Michael:

Screening microalgae strains for their productivity in methane following anaerobic digestion

http://www.sciencedirect.com/science/article/pii/S0306261913001694

Robert:

Hansen and Viamajala isolated three strains of algae from wastewater lagoons in Utah on
anaerobic digester effluent, and tested their growth on anaerobic digester effluent under varying
conditions. They found that growth rates and biomass production increased after a period of
adaptation to the effluent, as well as with supplementation of light, with the best growing strain
producing 2.71 g/L of biomass on average.
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Nutrient Utilization from Anaerobic Digester Effluent Through Algae Cultivation

http://digitalcommons.usu.edu/etd/671/

Researchers at University of Florida investigated the feasibility of anaerobic digestion for

organic waste treatment on space missions. They found conversion efficiencies of 75% and
85%, with residence times of 2-3 weeks, for various blends of organic waste, without need for
pH control.

Anaerobic Digestion for Reduction and Stabilization of Organic Solid Wastes During Space
Missions: Laboratory Studies

abe.ufl.edu/chyn/download/publications_dc/refereed/icesadlabdcrev1.pdf

Giani and everyone else:

Find and summarize more papers relevant to algae grown on wastewater, algae

oxygen production, and algae anaerobic digestion for methane production.

Methods

Results

Discussion & Conclusion

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Works Cited

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Works Cited Additional Information

Author Author’s Credentials URL’s

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Appendix A
Research Question Planning Notes
(​Education and Council 2012​)

Research Question:​ “Formulate a question that can be investigated within the scope of the
classroom, school laboratory, or field with available resources” (3a.1)

What are some uses algae could provide to a mars colonization effort?

Hypothesis & Prediction:​ “Frame an appropriate hypothesis (that is, a possible explanation that
predicts a particular and stable outcome) based on a model or theory.” (3a.2)

It may be possible for algae to produce a sustainable source of oxygen, glucose and methane for
a Martian colony.

Data:​ “What data are to be gathered” (3b.1)

- Growth rate of algae on wastewater

- Oxygen production of algae growth
- Methane production from algal biomass

Tools:​ “What tools are needed to do the data gathering?”(3b.2)

- Algae cultivation system

- Algal culture
- Clean room
- Wastewater/AD effluent
- Oxygen sensor
- Anaerobic digester
- Infared sensor for methane quantification

Measurements:​ How are measurements to be recorded? (3b.3)

- Nutrient in wastewater - manually ahead of time and afterwards

- Oxygen - automatically as it’s produced
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- Methane automatically as it’s produced

Quantity of Data​: “How much data are needed to produce reliable measurements” (3c.1)

- Look at literature

Data Limitations:​ “Consider any limitations on the precision of the data.” (3c.2)

Different growth conditions on mars?

Availability of nutrients and CO2 on mars.

Procedures:​ “Plan experimental or field-research procedures.” (3d.1)

- Look at literature

Independent & Dependent Variables:​ “Identify relevant independent and dependent variables”
(3d.1)

- Independent variables
- Nutrients
- CO2
- Light
- Dependent
- Growth rate
- Oxygen production
- Methane production

Controls: “​Identify the need for controls.” (3d.3)

None

Confounding Variables / Effects:​ “What are the possible confounding variables or effects?”
(3e.1)

Different conditions on mars. Locational variables. Contamination.

Control of Confounding Variables / Effects; “​Describe how the investigation’s design has
controlled for the possible confounding variables or effects.” (3e.2)
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- Look at literature.

REFERENCES:

Education, C. o. C. F. f. t. N. K.-S. and S. N. R. Council (2012). ​A Framework for K-12 Science Education:
Practices,

Crosscutting Concepts, and Core Ideas​, National Academies Press, Washington DC.

Appendix B
1
Research Question Analysis Notes
(​Education and Council 2012​)

SCIENCE & ENGINEERING PRACTICES #4

4. Analyzing and Interpreting Data (p62/pdfp 77)

You are going to prepare for your research by constructing sample data tables, charts
spreadsheets etc. and populate them with sample data that you “makeup.” Make two sets
of data one that proves your hypothesis and one that does not. Answer the questions below
using your “virtual sample” data. Place your data in Appendix D before you answer the
questions below.

4a.1 Systematically analyze the data from your research. In the space below, describe the salient
(​most noticeable or important​) patterns that you observed.

4a.2.1 Is your data consistent with an initial hypothesis or not?

1
Revised from the following word doc = “SEP Goals & Crosscuts ConceptMapsByRW 20160205.docx”
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4a2.2 Describe how you tested the data to determine if it was consistent with the initial
hypothesis or not.

• 4b1 Describe how you would recognize if your data was in conflict with the expectations you
had before your research began.

4b.2 Give an example of when your data was in conflict with the your expectations and describe
revisions you would make in the initial model to resolve this conflict.

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• 4c Use email or Google Drive or a flash drive to submit ALL of the following data types to
your teacher: spreadsheets, databases, tables, charts, graphs, statistics, mathematics, and
information and computer technology. When you meet with your teacher to review your work be
prepared to describe how you did each of the following. After you have submitted the data and
when you know, you are prepared to describe these items to your teacher, check off the
appropriate box in the Student Data Checklist below. The teacher will verify your checks
according to what you say during your interview using the Teacher Data Checklist below.

Student Data Checklist

Spreadsheet Databases Tables Charts Graphs Statistics Math Info &
(4c.1) (4c.2) (4c.3) (4c.4) (4c.5) (4c.6) (4c.7) Computer
Technology
(4c.8)
a. Collate
Data

b. Summarize
Data

c. Display
Data

d. Explore
Input
Output
Variables

e. Explore
Other
Variables

Teacher Data Checklist

Spreadsheet Databases Tables Charts Graphs Statistics Math Info &
(4c.1) (4c.2) (4c.3) (4c.4) (4c.5) (4c.6) (4c.7) Computer
Technology
(4c.8)

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f. Collate
Data

g. Summarize
Data

h. Display
Data

i. Explore
Input
Output
Variables

j. Explore
Other
Variables

4d.1 What conclusion can be inferred from your data set?

4d.2 Using appropriate grade-level mathematical and statistical techniques evaluate the strength
of the conclusion that you inferred from your data set.

4e.1 Recognize patterns in your data that suggest relationships worth investigating further and
list them below.

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4e.2 Describe at least one or more causal relationships you discovered in your research.

4e.2 Describe at least one or more correlational relationships you discovered in your research.

4e.2 Describe how you distinguished between the causal and correlational relationships
discovered in your work.

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4f.1.1 Draw a picture or diagram of at least one of the physical models you used or made in your
research.

4f.1.1 What data did you collect from the physical model(s) used or made in your research?

4f.2 Describe how you used the data collected from the physical model(s) you used in your
research to analyze the performance of your design under a range of conditions.

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Appendix C:
Specific Experimental Procedures

A.) Measure Nutrient and pH Content of A.D. effluent

1.) 1000 mL of anaerobic digester effluent was obtained from a local municipal waste
biogas facility
2.) 1 mL of this effluent was measured out for each of the following tests
3.) pH was measured using litmus strip test
4.) Nutrients were tested using the following chemicals and procedures
a.) Reagents: • Strong acid solution: An aliquot of 300 ml concentrated sulphuric
acid was slowly added to 600 ml distilled water. When cooled, 4 ml of nitric acid
was added and the mixture was diluted to 1 liter.
b.) Ammonium molybdate solution: It was prepared by dissolving 25 g Ammonium
molybdate in 500 ml distilled water.
c.) Stannous chloride solution: It was prepared by dissolving 2.5 g of stannous
chloride in 100 ml glycerol with continuous stirring to fasten the dissolution.
d.) Phenolphthalein indicator: It was prepared by dissolving 0.5 g of phenolphthalein
powder in 100 ml of 60% alcohol (Ethanol).
e.) Standard phosphate solution: It was prepared by mixing 1.436 g of potassium
hydrogen phosphate solution in 1 liter distilled water (100 ppm).
f.) Different dilutions (5, 10, 15, 20 and 25 ppm) were made from standard solution
by taking 5, 10, 15, 20 and 25 ml and diluted up to 100 ml with distilled water.
g.) Procedures:
i.) An aliquot of 50 ml of solution was taken in a flask and few drops of
phenolphthalein indicator were added into it. If pink color developed, small
amount of strong acid solution was added drop wise, just to discharge the
color. An aliquot 4 ml of ammonium molybdate was added slowly followed
by the addition of 4-5 drops of stannous chloride with through mixing after
each addition. Samples were left unshaken for 10 minutes at room
temperature for color development. 4.) The absorbance was measured at
610nm. Calculations: Standard curve was prepared against phosphate
concentration. The phosphate concentration of the sample was computed
by comparing with standard curve.
ii.) Nitrate-nitrogen (NO3-N): EPA method 4500 NO3-N was used to
determine nitrates in water (Standard Methods, 2005). Reagents: •Phenol

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disulphonic acid solution • Concentrated ammonia solution •

Standard solution of KNO3: It was prepared by dissolving 3.61 g of KNO3
in 500 ml distilled water (100 ppm).Different dilutions (0.5, 1, 1.5, 2 and
2.5 ppm) were made from standard solution by taking 0.5, 1, 1.5, 2 and
2.5 ml and diluted upto 100 ml with distilled water. Procedure: An aliquot
of 50 ml sample was taken in a china dish and evaporated on hot plate
until it become dry. Then 0.5 ml of phenol disulphonic acid was added to
the sides of china dish. It was cooled at room temperature and 6 to 8 ml
of conc. NH3 solution was added and mixed well. The sample was then
diluted upto 100 ml with distilled water and then absorbance was
measured at 410nm. Calculations: Standard curve was prepared against
nitrate concentration. The nitrate concentration of the sample was
computed by comparing it with standard curve.
iii.) Nitrite-nitrogen (NO2-N): 4500 NO2-N method was used to determine
nitrites in water sample (APHA, 2005). Reagents: • Buffer’s color reagent:
It was prepared by mixing 105 ml concentrated HCl, 5 g sulfanilamide and
0. 5g N (1-naphthyl) ethylene diamine dihydrogen chloride in 250 ml of
distilled water with constant stirring for dissolution. Then 136 g of sodium
acetate was added to this mixture with constant stirring. After dissolution,
the mixture was diluted upto 500 ml with distilled water. • Stock nitrite
solution: It was prepared by dissolving 0.5 g sodium nitrite in 1000ml of
distilled water (100 ppm).Different dilutions (0.2, 0.4, 0.6, 0.8. 1 and 2
ppm) were made from standard solution by taking 0.2, 0.4, 0.6, 0.8, 1 and
2 ml and diluted upto 100 ml with distilled water. Procedure: An aliquot of
50 ml of well filtered sample was taken in 100 ml Nessler’s tube and 2 ml
of buffer reagent was added into it and mixed thoroughly until the color
appears within 15 minutes. Then absorbance was measured at 540nm.
Calculations: Standard curve was prepared against nitrite concentration.
The nitrite concentration of the sample was computed by comparing it
with standard curve.

B.) Gas Exchange In Algae :Finding Optimal Amount Of CO2 For O2

Creation
● Have a 900mL controlled container filled with algae to be used for recording the
gas exchanging.

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● Nutrient medium will also be added to the container. (basal salt and 1g of
Nitrogen per liter) ​<- this should be done using anaerobic
● Essential lighting of ~130x10^15 to 140x10^15 of quanta sec/cm would also be
added.
● An opening for a steady flow of 100-105ml/min of air would flow into the
container with ~2-2.2% CO2
● Two electrodes for CO2 would be added into the nutrient medium from the gas
flow entrance and another at the gas exhaust port.
● Analyze the oxygen levels using a gas analyzer with a small 0.5mL sample
● With the base reading, change the CO2 levels in variations of 0.5% between
0.5% and 10% and record the oxygen levels.
● Record And Graph the relations between the percentage levels of CO2 and the
amount of Oxygen produced from the algae
● We also need to measure the algal biomass production rates for all of these set
ups

C.​) Environmental Impact On Oxygen Production

● Take three concealed water environments all with a dimension of 50x30x30cm.
Have two holes on either side of the controlled environment to help circulated the
environment when creating water velocity. ​< - can we get a diagram of this, so
we know what this set up looks like?
● Take each O2 level scenario and repeat experiment but this time have one of the
environments have inorganic CO2 within the water system ​<- this should be done
for all three environments, not just one. This can be done by a bubble stone, as
in an aquarium.
● Again take each of the three initial environments and change the water velocity to
0m/s 5m/s and 10m/s ​<- this flow differentiation should be done both for the
bubbled environment and for the non-bubbled environment

D.) Test nutrient removal and pH change by algae, using optimal growth conditions
1.) Set up a growth trial, using the optimal conditions from sections b and c
2.) Repeat steps 2-4 from section A using samples of post cultivation medium

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E.) Creating Hydrogen and Methane ​C. vulgaris

https://biotechnologyforbiofuels.biomedcentral.com/track/pdf/10.1186/1754-
6834-4-34?site=biotechnologyforbiofuels.biomedcentral.com

● Chlorella vulgaris​ will be used to produce both H2 and CH4

● Take both microalgal biomasses and place them in separate control bottles at
37C
● For each bottle, have the ratios of 25% biomass with 75% activated anaerobic
digester sludge, 50% of each, 75% biomass and 25% sludge, and 100% biomass
within a substrate.
● Record the production CH4 for both algae and their different biomasses.
● A controlled biomass environment is made in bottles for creating H2. No
inoculum is to be used in the process.
● All procedures will be done with “dark fermenting”, where the process of H2
creation will be done in a dark and anaerobic environment.
● Indirect photolysis of the biomasses will be obtained by hydrolyzing the
biomasses with lactic acid and with that should be the production of H2 through
photosynthesis.
● With the control environment created, lower the pH levels of the environment by
creating duplicate environments, but with a ratio of 20%, 40%, and 60% glucose
levels in the biomasses. ​<- how exactly is the glucose levels changed in the
biomass?
● Record your studies and adjust accordingly to maximize both H2 and CH4
productions.

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Appendix D:
Specific Experimental Data

A.) Measure Nutrient and pH Content of A.D. effluent

● Best rate of anaerobic digestion is when cell lipid content does not go over 40%
● Best growing strain producing 2.71 g/L of biomass
● Anaerobic digestion for organic waste treatment conversion efficiencies of 75%
and 85% which took 2-3 weeks
● Chemical content of nutrient and pH:
Optimal pH-- 7-8
Phosphorus-- .11 mg/L
Nitrite-- .1 mg/L
Nitrate-- .24 mg/L

B.) Gas Exchange In Algae :Finding Optimal Amount Of CO2 For O2

Creation

● Maximum output of photosynthesis was 1.5-2.5% by volume

● 2% CO2 flowing at 320 ml/minute, 3% CO2 at 135 ml/minute and 4% CO2 at 55
ml/minute yielded optimal CO2 concentrations

Oxygen Production
Flow Rate 1% CO2 2% CO2 3% CO2 4% CO2 5% CO2 6% CO2
m/s

0 m/s .096mg .096mg .096mg .096mg .096mg .096mg

5 m/s .098mg .1mg .102mg .105mg .111mg .109mg

10 m/s .12mg .132mg .136mg .14mg .087mg .085mg

C.​) Environmental Impact On Oxygen Production
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● Maximum O2 production was 2.43 g/L

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D.) Test nutrient removal and pH change by algae, using optimal growth
conditions

● Nutrient removal was .36 ml/h

​ nd ​D.tertiolecta
E.) Creating Hydrogen and Methane ​C. vulgaris a

● the process generated 46 ml of Hydrogen gas and 396 ml of Methane

● When used the same batch multiple times the Methane production rate improves
● If ammonia levels went up the production decreases
● Methane production: 351 ml produced at 25% biomass/75% activated anaerobic
digester sludge, 366 ml produced at 50% each, 297 ml produced at 75%f
biomass/25% activated anaerobic digester sludge, and 284 ml produced at 100%
biomass within a substrate
● Hydrogen production: 29ml produced at 20% glucose, 32 ml produced at 40%
glucose, and 37 ml produced at 60% glucose

For appendix D we first measured the nutrient and PH content of A.D effluent. For
anaerobic digestion the best rate was when the lipid content doesn't go over 40%. While the best
strain was producing 2.71 g/l biomass while the organic waste treatment conversion for
anaerobic digestion has an efficiency of 75% and 85% which took 2-3 weeks. The chemical
content of nutrients and PH optimally will be between 7 and 8. We focused on 3 elements in
particular nitrate(.24 mg/l), nitrite(.1 mg/l), and phosphorus(.11 mg/l). Next we focused on the
gas exchange in the algae and finding the optimal amount of CO2 and O2 creation. The max
output of photosynthesis was 1.5%-2.5% by volume. We had ​2% CO2 flowing at 320
ml/minute, 3% CO2 at 135 ml/minute and 4% CO2 at 55 ml/minute yielded optimal CO2
concentrations​. Next we zoomed in on O2 production. As we increased CO2 levels from
0.004%,2%,5%,10-70%. At 0m/s the O2 production is 0.3ml/min at 0.04%, 0.98ml/min at 2%,
3.03ml/min at 5%, 5.05ml/min at 10%, 4ml/min at 50%, 0.85ml/min at 60%, 0ml/min at 70%. At
5m/s O2 production is 0.56ml/min at 0.04%, 1.13ml/min at 2%, 3.18ml/min at 5%, 5.21ml/min
at 10%, 4.17ml/min at 50%, 0.96m/min at 60%, 0ml/min at 70%. At 10m/s )2 production was
0.72ml/min at 0.04%, 1.21ml/min at 2%, 3.3ml/min at 5%, 5.34ml/min at 10%, 4.29ml/min at
50%,1.03ml/min at 60%, 0ml/min at 70%. The maximum O2 production was 2.43 g/l. the
nutrition removal and PH change by algae with optimal levels was .36 ml/h for the nutrient
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removal. Then creating hydrogen and methane the process generated 46 ml of hydrogen gas and
396 ml of methane, though if ammonia levels start rising the production will decrease. Methane
had ​351 ml produced at 25% biomass/75% activated anaerobic digester sludge, 366 ml
produced at 50% each, 297 ml produced at 75%f biomass/25% activated anaerobic
digester sludge, and 284 ml produced at 100% biomass within a substrate. Where as
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hydrogen had 29ml produced at 20% glucose, 32 ml produced at 40% glucose, and 37
ml produced at 60% glucose.

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