This standard operating procedure document provides the safety guidelines for extracting oils from algae using n-Hexane solvent. It outlines the procedures for cell density measurement, algae harvesting, pump operation for fractionating the algae-hexane mixture, and evaporating the hexane to recover the algal oil. The document includes appendices with figures illustrating the equipment setup, a schematic of fluid flows through the process, and a lab safety map. Training and sign-off are required before using these extraction procedures.

1. Standard Operating Procedures for Algae
Based Oil Production
James Madison University
March 2015
Written by: Hannah Aloumouati, Victoria Foster, and Alexander MacFarlane
Advisor: Dr. Chris Bachmann, Ph. D

2. 1
Executive Summary
This report provides the standard operating procedures necessary for the operation of a system
used to extract oils from an algae species by using recoverable n-Hexane throughout the process.
These procedures meet the safety guidelines for the handling of solvent in the lab as set forth by
the James Madison University Environmental Health Coordinator. All figures, the Safety Data
Sheets (SDS) for n-Hexane, and a list of important safety locations in the Alternative Fuel Vehicle
Lab are provided in the appendices. It is mandatory to read and understand these standard operating
procedures to ensure safety and accuracy in results.

3. 2
Table of Contents
Overview of Standard Operating Procedures ........................................................................................3
Purpose:.........................................................................................................................................3
Physical & Chemical Properties/Definition of Chemical Group .............................................................3
Protocol/Procedure for Pump Actions and Reclamation Device .............................................................4
Cell Density ...................................................................................................................................4
Harvesting......................................................................................................................................4
Preparation of Reclamation Device ..................................................................................................5
Fractionation of Output (follow only if reclamation is not successful).................................................6
Evaporation and Condensation.........................................................................................................6
Post Evaporation.............................................................................................................................6
Appendix B......................................................................................................................................14
Appendix C......................................................................................................................................16

4. 3
Overview of Standard Operating Procedures
Department: James Madison University – Alternative Fuels Vehicle Lab
SOP Creation: 2-25-2015
SOP Approval:
Location covered by this SOP: JMU-Alternative Fuel Vehicle Lab
1595 S. Main Street MSC 5401
Harrisonburg, VA 22801
(540)-568-3329
Type of SOP: Process Hazardous Chemical Hazardous Class
Purpose:
Continuously evaporate n-Hexane from solution and recover by condensation.
Physical& Chemical Properties/Definitionof ChemicalGroup
CAS#: 110-54-3
Class: 3
Form (physical state): liquid & vapor
Color: transparent, colorless
Potential Hazards/Toxicity: Flammable liquid and vapor. The acute toxicity of n-Hexane is low.
Danger of serious damage to health by prolonged exposure through inhalation. Breathing vapors
may cause drowsiness and dizziness. Causes eye and skin irritation. Possible risk of impaired
fertility. Aspiration hazard if swallowed. Can enter lungs and cause damage. TARGET ORGANS:
blood, central nervous system, liver, respiratory system, eyes and skin. See the SDS provided in
the appendix for more toxicity information or MSD of n-Hexane in flammable cabinet.
Personal Protective Equipment (PPE)
Use only under a chemical fume hood. Have emergency contact information in reach.
Eyes: Safety glasses equipped with side shield are required as a minimum protection in the
industrial setting. Chemical goggles should be worn during transfer operations or when there is a
likelihood of misting, splashing, or spraying of this material. A suitable emergency eye wash water
and safety shower should be located near the work station.
Skin: Avoid skin contact. Use heavy duty gloves that are chemical resistant such as Vilton® or
nitrile rubber. Wash hands with plenty of mild soap and water before eating, drinking, smoking,
use of toilet facilities or leaving the work setting.
Clothing: Wear appropriate protective clothing to prevent skin exposure, closed toed shoes, hair
tied back, no dangling jewelry, and no loose clothing.

5. 4
Protocol/Procedure for Pump Actions and Reclamation Device
Proper preparation includes prior knowledge of the SDS for all chemicals used as well as lab safety
certification from lab supervisor.
Cell Density
1. All active participants wear PPE, have hair tied back and closed toed shoes.
2. Clean the microscope, hemocytometer, and the coverslip.
3. Place coverslip over hemocytometer then with a micropipette place 10uL of algae sample
on hemocytometer until the grid pattern becomes full.
4. Focus the microscope at 4x course fine, then 10x fine, and finally 40x fine until the grid
pattern on the hemocytometer becomes clear.
5. Choose all the blocks to achieve an evenly distributed spread and count the amount of algae
cells in the blocks.
6. Determine the volume of the sample to obtain the cell density.
7. Clean slide and return to storage location.
8. Find volume of 10uL of algae
*Measured cell density=(average cells per small square X dilution factor)/(volume of a
small square (mL))
*The volume of a small square is specific to the hemocytometer. It is calculated by multiplying
the width by the height (which are the same – usually 1mm each) by the depth (usually 0.1mm) of
a small square. In the most
Harvesting
1. Before entering the lab zone, put on protective gloves, eye protection, have hair tied back,
closed toed shoes and no loose clothing.
2. Open fume hood from the front access panel and secure it using the provided bungee
connector to the above light fixture, plug in yellow power cord to outlet.
3. Open vent panel that leads outside with the attached purple lever (refer to arrows marked
on the side of the vent for open and closed position).
4. Turn on the fume hood exhaust fan using the power control unit.
5. Fill bucket labeled “Primer Input” with desired amount of saltwater and record volume in
lab notebook (Salinity for 5 gallons of water, add 673.73 grams salt).
6. Fill bucket labeled “Measured Input” with desired amount of saltwater/algae mix and
record volume in lab notebook.
7. Use the protective rubber container to carry n-Hexane from the flammable storage cabinet
to the fume hood.
8. With the Fume Hood operating, measure amount of n-Hexane in to glass beaker labeled
“n-Hexane Measured Input” and record volume in lab notebook.
9. Keep n-Hexane input covered with foil to prevent any contact and stabilize hose from pump
in the beaker.
10. Return the n-Hexane container safely back into the flammable cabinet for storage.
11. Place container labeled “Measured n-Hexane/Saltwater Output” into fume hood.

6. 5
12. Attach an energy reader (Kill A Watt Energy Monitor) to power cord of solvent mixing
pump to record revolving current (Optional if viable).
13. Pour approximately 2 L of water into primer input. The machine has to be primed in order
to begin the flow of liquids.
14. The device will reach a steady state after there is a continuous flow in the pump from the
input to output; aka “being primed”.
15. Set the valves on the pump to the appropriate settings (see Figure 3 in Appendix A).
16. Turn on pump and run the “Primer Saltwater Input” (see Figure in Appendix A) through
the device. *Make sure the outflow valve is set to “Primer Saltwater Output”.
17. After primer steady state is established, begin algae inflow.
18. Flip the inflow valve (see Figure 3 in Appendix A) to begin extracting from the “Input”
container (the arrows on the valves should direct the mixture path).
19. Measure the time frame it takes for algae to flow from the input to the output. Record data
(notice the color changes to confirm the algae flow—clear liquid to green).
20. Make sure there is a continuous flow of algae through the pump.
21. After algae steady state is established, begin n-Hexane inflow.
22. Turn red control flow valve (see Figure 3 in Appendix A) to being extracting n-Hexane
from the “n-Hexane Input”. *The red control flow valve controls the amount of n-Hexane
into the system. *Clockwise closes.
23. Refer to the time frame recorded previously to determine when n-Hexane will reach output.
Once that time is reached, switch the outflow valve to begin flow into the “Measured n-
Hexane/Saltwater Output” protective container (the arrows on the valves should direct the
mixture path).
24. Once desired volume is reached, stop extracting n-Hexane and algae and switch the inflow
valves back to the primer.
25. Make sure “n-Hexane/Primer Output” tube on the pump is in the “n-Hexane Clean Out”
carboy in the fume hood.
26. Refer to the time frame recorded previously to determine when the primer will reach the
output. Once that time is reached, switch output to “n-Hexane Clean Out”.
27. For two minutes allow the device to circulate with the primer to clear out the remainder of
the n-Hexane solvent from the pump. Then turn the pump off.
28. When the experimenting is done, run water through the entire system to cleanse the pump.
29. Cap off the “Measured n-Hexane/Saltwater Output” carboy for later use *explained in the
Appendix C.
30. Allow the n-Hexane in the “n-Hexane Clean Out” carboy to evaporate off while fume hood
is still operating.
31. Turn off fume hood after evaporation is complete and cap carboy.
Preparation of Reclamation Device
1. Open fume hood front.
2. Turn on fume hood exhaust fan using the power control unit located in front of the fume
hood.
3. Place “n-Hexane/Algae Mix” output unit into fume hood.
4. Attach hot and cold water supply tubing to evaporator and condenser respectively. Turn on
water heater and allow temperature of hot water supply to reach operating temperature
before turning on heating fluid pump. (see Figure 8 and Figure 9 in Appendix A)

7. 6
5. Turn on heating fluid pump.
6. Allow the evaporator to fill with hot water. Begin cycling heating fluid and for the
temperature of the unit to stabilize.
7. Prepare cold water supply by filling a five gallon bucket with 20 lbs of ice and adding water
to an appropriate level before turning on pump.
8. Turn on cooling fluid pump.
9. Allow condenser coil to begin cycling cooling fluid and for temperature to stabilize.
10. Place appropriate collection vessels and outlets of both the condenser and evaporator and
ensure that the valve of the evaporator exit is closed.
11. Do all PPE as stated previously.
12. Obtain the “Measured n-Hexane/Saltwater Output” carboy in fume hood.
13. Mix the Oil/n-Hexane miscella until homogenous in fume with glass rod.
14. With the carboy valve closed, connect the carboy to the miscella inlet.
Fractionation of Output (follow only if reclamation is not successful)
1. Turn on fume hood
2. While the “n-Hexane/Saltwater Output” container remains in the fume hood, allow
separation for 24 hours to achieve distinct layers.
3. After waiting 24 hours weigh glass petri dish and record its mass.
4. Using the fume hood, collect sample of desired volume from the n-Hexane layer using a
glass pipette and spread on glass petri dish. Reweigh the petri dish. Repeat this three times.
5. Keeping the petri dish in the fume hood allow the sample to dry for 24 hours to evaporate
n-Hexane out of the sample. Reweigh the sample.
Evaporation and Condensation
1. Continue with the evaporation and condensation procedure only after the completion of the
Reclamation Preparation phase as dictated above.
2. Open the condenser outlet valve (see Figure 7 in Appendix A, down open/ up close).
3. Slowly open the carboy valve until desired flow is achieved into miscella valve
4. Open the evaporator outlet slowly.
5. Allow evaporation to occur completely. Evaporation is complete when the combined
volumes (or weight) of Oil and n-Hexane in the collection vessels is equal to the initial
miscella volume (or weight) added to the carboy or once there is no output from either the
condenser or evaporator valves.
6. Close the carboy.
Post Evaporation
1. Turn off the water heater.
2. Turn off the water supply to both the evaporator and the condenser.
3. Perform any necessary measurements or analysis of the Algal Oil and n-Hexane, use and
necessary glassware to collect oil reclaimed and store of the two substances according to
safety regulations.

8. 7
4. Once the evaporator has cooled, remove the water supply lines from the system and drain
all water.
5. Allow all vapors to be evacuated from the system before closing the evaporator inlet/outlet
valves.
6. Allow all vessels to dry completely before removing from the fume hood.
7. Clean all glassware thoroughly and store tools appropriately.
8. Remove evaporator from under the fume hood and store.
9. Turn off fume hood and shut the panel connected to fan.
10. Be sure all Hexane is in the flammable safety cabinet.
NOTE: SOP derivation requires prior approval and inspection from the Safety Marshal.
* When the machine is not being used for more than two weeks, run oil through all inputs until oil
flows out of the output. Then turn off pump and proceed to raise tubes above the output and input
height and secure tubes in order to prevent oil leakage.
Documentation of Training: (signatures required)
 Prior to conducting any work with n-Hexane, the principle investigator must provide
training to his/her laboratory personnel to the hazards involved in working with this
substance, work area decontamination, and emergency procedures.
 The Principle Investigator must ensure that his/her laboratory personnel have attended
appropriate laboratory safety training or refresher training within the last one year.
 The Principle Investigator must provide his/her personnel copy of his SOP and a copy of
the chemical SDS provided by the manufacturer.
I have read and understand the content of this SOP:

9. 8
NAME SIGNATURE DATE

10. 9
Figure 1:This is an image that displays the set up
of the pump arrangement while algae-oil
extraction is occurring. Red tape indicates the
inputs and outputs of the hexane fluids, green
represents the algae input, and blue represents the
primer.
Appendix A
Figure 2:This image is a closer view of Figure 1. In this
photo, the direction of the flows through the pump is
dictated by the yellow handles. In this specific picture, all
the flows are restrictedbased on the arrows located on the
yellow handles.

11. 10
1
3
2
4
Figure 3:This image is a top-down view of the entire pump with
all the labeled tubes, inputs, outputs, and flow directions. The
numbers correspond to the schematic.
Figure 4: This shows the pump with some of the fume hood in the view.
Hexane input comes from the beaker in the fume hood and constrained
by the wooden block for support, while the hexane output is going back
into the fume hood for safety reasons.
Recirculationvalve

12. 11
Figure 6: This is the tub of the algae being used to conduct this
experiment. Many aeration tubes, a buoy for the dechlorinated
water top off system, and a smaller algae sample is displayed in
this photo.
Figure 5: This view of the experimental setup shows the
buckets labeled with eachtube with a corresponding input
or output bucket.

13. 12
Figure 7: This is a full photo of the fume hood. As shown, to open the fume
hood a bungee is used to tie open the window, and the air is sucked from the
fume hood to the outside of the lab facility while the fume hood is on. All the
hexane products and inputs are located in there.
Hexane input
Figure 8: This image displays the entire
contraption and parts of the tubing for shellside
fluids.
Bungee Front AccessVent Panel
Power Control
Heating Fluid Outlet
Evaporator Valve
Vent lever

14. 13
Figure 9: This image displays the condenser
outlets and inlets as well asthe miscella inlet.
All are marked accordingly.
Figure 10: This view is a close up of the base
of the contraption.
Miscella Inlet (inletofalgal oil and hexane mixture)
Condenser InletCondenser Outlet
Heating Fluid Inlet

15. 14
Appendix B
Figure 11: This is a schematic showing the directional flow for each of the contents involved in algae
oil extraction process using the pump. The red line represents hexane contents,the green line represents
algae contents, and the blue line represents the primer contents. The arrows direct the flow of the fluid
through the entire process. Ultimately, after the primer runs through the process, the algae and hexane
flow will be united to make cell separation occur. The schematic just shows the individual flow for each
fluid.

16. 15
Exit
Exit Garage Exit 1 Garage Exit 2 Exit
Algae
Shelf
TopOffSystem
Eyewash&Shower
FumeHood
GlasswareCleaningStationDesk
OtherEquipment
Figure 12: This is a schematic of the Alternative Fuels Vehicle Lab Algae set up. The red
circles indicate a fire extinguisher. This image is not a representation of the actual scale of the
lab.
Supply
Cabinet
Flammable Cabinet#1
SDS Files
Flammable Cabinet#2
SDS Files

17. 16
Appendix C

18. 17

19. 18

20. 19

21. 20

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24. 23