Algae Capstone S.O.P.

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

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.

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Table of ContentsOverview 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

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Overview of Standard Operating Procedures

Department: James Madison University – Alternative Fuels Vehicle Lab SOP Creation: 2-25-2015SOP 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/Definition of Chemical Group CAS#: 110-54-3Class: 3

Form (physical state): liquid & vaporColor: transparent, colorlessPotential 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.

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Protocol/Procedure for Pump Actions and Reclamation DeviceProper 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.

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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.

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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)

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 hood2. 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.

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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.

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.

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I have read and understand the content of this SOP:NAME SIGNATURE DATE

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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

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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 restricted based on the arrows located on the yellow handles.

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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.

Recirculation valve

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.

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Figure 5: This view of the experimental setup shows the buckets labeled with each tube with a corresponding input or output bucket.  

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.

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Bungee Front AccessVent Panel

Power Control

Heating Fluid Outlet

Evaporator Valve

Vent lever

Figure 9: This image displays the condenser outlets and inlets as well as the miscella inlet. All are marked accordingly.

Figure 10: This view is a close up of the base of the contraption.

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Miscella Inlet (inlet of algal oil and hexane mixture)

Condenser InletCondenser Outlet

Heating Fluid Inlet

Appendix B

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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.

Exit

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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.

Supp

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Flammable Cabinet #1SDS Files

Flammable Cabinet #2SDS Files

Exit Garage Exit 1 Garage Exit 2 Exit

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Appendix C

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