Methods
PROCEDURES for BUILDING THE PHOTOAUTOTROPHIC VESSELS & BIOREACTOR
The bioreactor and vessels will be constructed at home under the direct supervision of a parent.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientists, and parents.
3. Review Occupational Safety and Health Administration (OSHA) documents regarding safe practices with the use of tools (www.osha.gov) with my designated supervisor.
4. Purchase all supplies and safety equipment.
5. With adult supervision, prepare to build by putting on safety goggles, gloves, respirator mask, and lab apron in a workshop that is in a well ventilated area.
DESIGNING AND CONSTRUCTION OF THE VESSELS BY DRAWING NUMBER DESIGNING VESSELS:
1. The vessels were designed using AutoCAD. Eight separate drawings (EV-002 through EV-010) were created for each part. Three drawings (EVA-002 through EVA-004) were created to assemble the vessels.
2. I reviewed the plans with my designated supervisor for approval.
3. I sent drawings EV-002 through EV-010 to Florida Machinery to machine the parts.
4. Once the parts were machined, I cleaned and deburred all parts and assembled the vessels.
5. I utilized all safety equipment including safety goggles, gloves, respirator mask, and lab apron.
6. I put the vessels together in a well-ventilated area with my supervisor.
CONSTRUCTING THE VESSELS:
1. The vessels were constructed under the supervision of my adult supervisor and wearing all safety equipment including safety goggles and lab apron.
2. Glue the 2.54cm x 30.3cm diameter grey PVC (EV008) to each end of the 15.24cm diameter x 61cm clear PVC pipe and 19cm diameter.
3. Glue the 2.54cm x 30.3cm diameter grey PVC (EV009) to each end of the 15.24cm diameter x 61cm clear PVC pipe and 19cm diameter.
4. Glue (EV010) to (EV009).
5. Glue both .64cm x 5.1cm x 55.9cm clear PVC (EV005) inside the product created in step #1 180˚ apart. Glue 3.8cm diameter x 10.2cm PVC rod (EV003) to the 2.54cm x 13.3 diameter grey PVC (EV002).
6. Glue 3.8cm diameter x 7.6cm PVC rod (EV004) to the 2.54cm x 13.3 diameter grey PVC (EV002).
7. Place 7.6cm diameter rubber gasket to the product created in step #2 and #3.
8. Cut water fill pipe into 1.26 cm lengths to create aerator.
9. Silicone airstone to 1.26 cm fill pipe.
10. Tighten airstone assemble to EV008.
11. Bolt the product created in step #3 and step #4 to the product created in step #1 using 8 - ¼/20 x ¾ hex head bolts.
12. Teflon tape the icemaker tee valve to the drive end of the vessel.
13. Repeat steps #1 through #12 twice to create second and third vessel.
CONSTRUCTING of BIOREACTOR:
1. Designed bioreactor on AutoCAD producing drawing EV-0001. Obtained approval of drawing from my designated supervisor.
2. Purchase all supplies and safety equipment.
3. Under the direct supervision of my adult supervisor, prepare to build by putting on safety goggles, gloves, and lab apron in workshop.
4. With adult assistance, cut the particle board into two 1.3cm x 48cm x 71cm long using a hand saw.
5. With adult assistance, cut seven support brackets out of 1.27cm x 5.08 x 71cm plastic trim board using a miter saw.
6. Attach support brackets to both sides of particle board and screw together.
7. Cut 2.54cm diameter slot x 7cm long in three places.
8. Cut first slot 12.7cm, second slot 37cm, and third slot 61cm along 71cm side of bioreactor.
9. Install three on/off dimmer switches to the front panel of bioreactor and wire to each Motor and main power supply terminal block.
ASSEMBLING PHOTOAUTOTROPHIC VESSELS INTO BIOREACTOR
1. Under the direct supervision of my adult supervisor, prepare to assemble the vessels in the bioreactor by putting on safety goggles, gloves, and lab apron in workshop
2. Attach one #25 sprocket 24 tooth to drive end of each vessel (total of 3).
3. Teflon tape air hydraulic rotary union and attach to both shaft ends of each vessel (total of 3).
4. Place each vessel into a slot in the bioreactor (total of 3).
5. Install #25 sprock 12 tooth to 1.25hp gear motor.
6. Bolt #6 x 1” screw through bioreactor side wall into 1.25hp gear motor and attach #25 chain with #25 masterlink (total of 3).
7. Wire gear motors to 120 volt terminal block.
8. Wire neutral lead of gear motors to speed control pots.
9. Wire 120 volt terminal block to three prong plug.
10. Attach .32cm flex hose to air hydraulic rotary unions.
11. Repeat step #10 six times – twice for each vessel.
12. Draw vacuum in outer chamber of vessel and close off tee valve.
13. Insert filler tube from Helium supply to tee valve and open tee valve and fill to atmospheric pressure. Close tee valve.
14. Repeat #13 using Neon.
15. Label each vessel, Neon, Helium, natural.
PROCEDURES for BUILDING NUTRIENT CONTROL CHAMBER (NCC)
Under direct supervision of my adult supervisor and wearing all safety equipment including safety goggles, gloves, and lab apron, I installed the two aquarium pumps on to the pine board with 4 drywall screws.
1. With adult supervision, cut particle board into 2 pieces -1.2cm x 91cm x 61cm - using electric handsaw.
2. Cut particle board into 2 pieces -1.2cm x 66cm x 61cm - using electric handsaw.
3. Cut four pieces of pine board - 1.27cm x 5cm x 91cm.
4. Cut four pieces of pine board - 1.27cm x 5cm x 48cm.
5. Cut two acrylic sheet - .56cm x 61cm x 89cm.
6. Working in a well ventilated area and using a respirator mask, glue CPVC pipe, caps, and tee together and drill 4 holes for flex tubing insertion. Take the .32cm flex tubing and plumb from aquarium pumps to CPVC pipe assembly.
With adult assistance, drill 2 sets of holes (1.75cm) into box and glue
CPVC union and reducing bushing to box.
10. With adult assistance, drill 1 hole (2.06cm) into box and glue valve and reducing bushing to box.
11. With adult assistance, drill 1.27cm hole and install electrical cord.
Connect a clear PVC hose (.9525cm x 45.72 cm) to each port on the outside of the chamber.
Attach each hose end from the NCC to each end of the shaft on the end
caps of each vessel.
Screw four casters to bottom of NCC.
PROCEDURES FOR ASSEMBLING PHOTOAUTOTROPIC
BIOREACTOR and NUTRIENT CONTROL CHAMBER (NCC)
1. Under the direct supervision of my adult supervisor, I prepared to build by putting on safety goggles, gloves, and lab apron in workshop.
2. Place bioreactor on top of NCC and connect a 1.3cm flex hosing from the bioreactors output and input ports to the NCC output and input ports.
PROCEDURES for BUILDING THE LIPID EXTRACTOR/TRANSESTERIFICATION CHAMBER
(“CELLULOSE BLASTER”)
1. Under the direct supervision of my adult supervisor, I prepared to build by putting on safety goggles, gloves, and lab apron in workshop.
2. Screw together 3/8npt brass fitting into pressure vessel.
3. Screw together ½npt close nipple into ½npt tee fitting.
4. Screw #2 to #1.
5. Screw together 1/2npt close nipple to gas value.
6. Screw #4 to #3.
7. Screw together 200 pressure gage to reducing bushing 1/4npt to ½npt.
8. Screw #6 to #2.
9. Slide ½ high pressure rubber hose to 1/2npt x 7.6cm nipple.
10. Attach 3 hose clamps to high pressure hose.
11. Insert EV A004 drawing to #9 and attach three hose clamps to high pressure hose.
12. Attach ring terminals from ultrasonic transducer and attach with #8 screws to EV
A004 on both ends.
The following apparatuses were built for starting the algae cultures:
PROCEDURES for BUILDING PHOTOBIORECTOR
The photo-bioreactor will be constructed at home under the supervision of a parent.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientist, and parents.
Review Occupational Safety and Health Administration (OSHA) documents regarding safe practices with the use of tools (www.osha.gov) with my adult supervisor.
Designed bioreactor on AutoCAD producing drawing EV-0000. Obtained
approval of drawing from designated supervisor.
5. Purchase all supplies and safety equipment.
Under the direct supervision of my adult supervisor, prepare to build by putting on safety goggles, gloves, and lab apron in workshop.
With adult assistance, cut the particle board into four 2.54cm x 15.24cm long slot using a hand saw.
With adult assistance, cut four support brackets out of 1.27cm x 5.08 x
68cm plastic trim board using a miter saw.
9. Attach support brackets to both sides of particle board and screw together.
10. Cover side surfaces of the particle board circles with silver duct tape.
11. Cut 35.5cm diameter concrete cardboard form into 28cm length tube.
12. Attach concrete tube to support brackets in four places using drywall screws.
13. Cut reflective shield to 28cm in length and glue to the inside of the concrete tube.
PROCEDURES for BUILDING INCUBATORS
The incubators and vessels will be constructed at home under the direct supervision of a parent.
1. Read 2011-2012 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor,
qualified scientists, and parents.
3. Review Occupational Safety and Health Administration (OSHA) documents
regarding safe practices with the use of tools (www.osha.gov) with my designated
supervisor.
4. Purchase all supplies and safety equipment.
5. With adult supervision, prepare to build by putting on safety goggles, gloves,
respirator mask and lab apron in a workshop that is in a well ventilated area.
CONSTRUCTION OF THE INCUBATORS
1. Glue the 7.62cm diameter x 5.08cm white PVC pipe to the 3.81cm reducing bushing.
2. Glue the 3.81cm diameter x 2.54cm white PVC pipe to the reducing bushing and PVC cross.
Glue the 3.81cm to 2.54cm reducing bushing to the end of the PVC cross.
4. Glue 2.69cm PVC pipe to the 3.81cm bushing.
5. Glue the aquarium pumps to the PVC pipe.
6. Glue the 3.81cm x 1.27cm reducing bushing to the PVC cross.
7. File down lip on reducing bushing.
8. Glue the 22.7cm PVC pipe to the 1.27cm threaded bushing.
9. Glue the 22.7 cm PVC pipe to the 1.27cm tee.
10. Glue the 1.27cm tee to the reducing bushing.
11. Glue the 2.54cm x 1.27cm PVC pipe to tee.
12. Glue the elbow to the 2.54cm x 1.27cm PVC pipe.
13. Thread pressure gauge to end of PVC elbow for pressurized vessel only.
14. Glue 3.17cm x1.27cm PVC pipe to the tee.
15. Drill four .48cm holes on a 45˚ angle into the 3.17cm x 1.27cm PVC pipe.
16. Glue check values into each of the four holes.
17. Connect each check values to the aquarium pumps with .32cm clear hose.
18. Drill a .64cm hole into the 1.27cm cap.
19. Cut inner tube valve stem and glue it into the 1.27cm cap.
20. Glue the 1.27cm cap to the 3.17cm x 1.27cm PVC pipe.
21. Drill .95cm hole into the 7.62cm reducing bushing.
22. Teflon tape the icemaker tee valve to the 7.62cm reducing bushing.
23. Attach 25.4cm x .32cm clear hose to icemaker tee valve.
24. Teflon tape 1.27cm plug and place into 1.27 elbow and tighten to create the incubator.
25. Repeat step #14 through #25 to create the second incubator.
ASSEMBLING SPARGERS IN INCUBATORS
1. Under the supervision of my adult supervisor and wearing all safety equipment including safety goggles and lab apron, I cut water fill pipe into 45.7cm lengths.
At the bulb end, drill .32cm diameter hole through the blub at 90˚ intervals creating four holes.
Working in a well-ventilated area wearing a respirator mask, glue 10.2cm coffee stirrers into each hole using Gorilla glue.
Once glue dries, cut the coffee stirrers down to 3.18cm lengths using a ruler and scissor.
5. Slide sparger into hose bib.
Place the incubator on top of the test tube and tighten with hose clamp to create the vessel.
PROCEDURES FOR GROWING INTIAL ALGAE CULTURE
All algae will be grown and tested at the school laboratory under direct supervision of my designated supervisor.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientist, and parents.
3. Order Spirogyra Algae Cat # 86 V 0650 from Ward’s Natural Science, P.O. Box 92912, Rochester, N.Y. 14692. The ATCC classifies Spirogyra as a BSL- 1 organism. The algae are stored in its original shipping container until separated into test tube solution.
4. Prior to testing, my supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment and algae.
All testing will be conducted in a school laboratory under the direct supervision of my designated supervisor.
While working with the algae, I will be wearing safety goggles, gloves, a protective lab coat, and a respirator mask.
7. Under the direct supervision of my designated supervisor, clean one test tube thoroughly, rinse with distilled water, and sterilize in pressure chamber.
8. Fill test tube with 1250ml of distilled water.
9. Wearing goggles, gloves, mask, and apron, add 250ml of concentrated Spirogyra biomass to the test tube from common culture.
10. Turn on each air pump to make sure sparger works. (Run for a full week before adding algae.)
11. Leave all air pumps on during the entire experiment to agitate algae.
12. Cultivate for 4 weeks until algae biomass is sufficient for transfer into vessels in photoautotrophic bioreactors.
PROCEDURES FOR GROWING and TESTING ALGAE
IN VESSELS IN PHOTOAUTOTROPHIC BIOREACTORS
All algae will be grown and tested at the school laboratory under direct supervision of my designated supervisor teacher.
1. Read 2012-2013 ISEF Rules and Guidelines.
Complete required ISEF forms and obtained signatures from designated
supervisor, qualified scientist, and parents.
Order iron powder, -100/+350 Mesh and Sodium Carbonate from MiniScience, Inc. 1059 Main Ave Clifton, NJ 07011. They will be stored in their original containers in a cool, dry, locked storage room until used in the experiment.
Purchase Rhino Power Carbon Dioxide 20oz. tank from Lowes.
Review the health hazard information (MSDS) for Iron powder with my designated supervisor. Iron powder has a hazardous level of 2, flammability level of 1, and reactivity of 1. Sodium Carbonate has a hazardous level of 3, flammability level of 1, and reactivity of 1. Safety precautions include avoiding contact with eyes, skin and clothing. I must wash thoroughly after handling. I must avoid breathing the dust and keep the container closed. The iron powder supplement and Bicarbonate will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the chemicals. I will wash my hands thoroughly after handling the Iron powder, Sodium Carbonate and algae.
Reviewed the health hazard information (MSDS) for Carbon Dioxide canister. CO₂ has a hazardous level of 1, flammability of 0 and reactivity of 0. The CO₂ will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the CO₂. I will wash my hands thoroughly after handling the CO₂.
Prior to testing, my supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment, test kit, algae and iron powder.
All testing will be conducted in a school laboratory under the direct supervision of my designated supervisor.
While working with the iron powder and algae, I will be wearing safety goggles,
gloves, a protective lab coat, and a respirator mask.
10. Split 750ml algae biomass equally into each of the three vessels.
Add 1250ml distilled water to each of the three vessels.
Add 3 grams of HCO₃ into each of the three vessels.
Attach nutrient control chamber (NCC) to each side of vessel and place in respective bioreactor.
With assistance from my supervisor, add 660ppms of CO₂ to NCC for air agitation into the vessels. Agitate for one hour.
Wearing all safety equipment and under direct supervision by my designated supervisor, stop gear motors and draw samples from each vessel to conduct cell count test utilizing hemacytometer and 400x microscope and a pH test with pH meter. I will remove a small amount of algae solution from each vessel through the tee value into a prelabeled sterilized test tube.
With assistance from my supervisor, using a sterilized eye dropper, place a small amount of the algae solution on the hemacytometer counting chamber. Dilute sample so the cells do not overlap on the Neubauer ruling grid.
Systematically count the cells in selected squares so that the total count is 100 cells or more.
Record cell count and pH reading for all three vessels.
Add in the required Na₂CO₃ to the testing sample. The amount of Na₂CO₃ added each week was determined by the pH readings. The goal was to maintain the solution at the optimal pH level of 8.2 to 8.5.
Return sample back to incubators and restart gear motors. Sterilize the hemacytometer and pH meter thoroughly. Wash hands thoroughly.
21. Each bioreactor shall be exposed to the natural light provided by the sun from
sunrise to sunset during the entire testing.
22. Under direct supervision by my designated supervisor, take weekly readings of CO₂
ppms in the NCC for 12 weeks and record data.
23. With my designated supervisor, conduct weekly cell count tests on each vessel
utilizing the hemacytometer for twelve weeks and record data.
24. With my designated supervisor, take weekly pH readings for twelve weeks and
record data for each vessel.
25. Return algae sample mixed with the required amount of NA₂CO₃ to incubators after
each cell count test and sterilize hemacytometer and test tube.
26. At the end of week twelve, terminate all CO₂ infusion to the vessels and add 1 gram of Fe₃ and 2 grams Na₂CO₃ to each vessel to promote lipid growth and flocculation.
PROCEDURES FOR FLOCCULATION
Prior to flocculation, my designated supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment and algae biomass.
All flocculation processes will be conducted in a school laboratory under the direct supervision of my designated supervisor. The laboratory is well-ventilated.
While working with the algae biomass, I will be wearing safety goggles, gloves, a protective lab coat, and a respirator mask.
With assistance from my supervisor, transfer all algae solution from each vessel into pre-labeled test tubes for flocculation.
5. Let algae biomass settle for 24 hours.
6. With my supervisor, record sedimentation volume using gravimetric analysis for each.
7. Wearing all safety equipment including safety goggles, gloves, respirator mask, and
lab apron, dewater each test tube to sedimentary level using a 60ml sterile syringe.
8. Place excess water in incubator samples.
9. With assistance from my supervisor, repeat step #4 through 8 for each vessel and record data, ensuring that each vessel’s algae slurry remains separate and clearly marked.
10. Wearing all safety equipment and under direct supervision by my supervisor, pour each flocculated algae slurry over the 7.6cm diameter N45 Neodymium magnet into a larger beaker attracting all the Fe² onto the magnet resulting in a clean biomass.
11. Repeat step#10 for each vessels slurry, ensuring that each vessels algae slurry
remains separate and clearly marked.
12. Use concentrated algae slurry biomass for the lipid extraction process.
PROCEDURES FOR EXTRACTING LIPIDS UTILIZING “CELLULOSE BLASTER”
All extracting procedures were done under the direct supervision of my designated supervisor in the school lab. All safety equipment including gloves, safety goggles, ventilation mask and apron were worn during extraction procedures.
With assistance from my supervisor, each vessel’s slurry was weight in labeled beakers on a gram scale. Ten percent of each vessels algae slurry was calculated and recorded.
Each slurry received 10% of their biomass weight in NaCl. The NaCl was vigorously mixed into each slurry.
Place the slurry into the “cellulose blaster’s” canister and assemble ultrasonic unit ensuring unit is tightly sealed.
Charge canister to 147 psi (10 bars) and close gas valve.
Let solution soak in osmotic bath for 1 hour.
Attach collection vessel to cellulose blaster.
Place canister upright and turn on ultrasonic unit.
Release pressure allowing solution to evacuate canister into collection vessel. Once all solution has passed through ultrasonic orifice close gas valve.
Collect biomass from collection vessel and place in vacuum chamber.
Attach hand held vacuum pump to port on lid of chamber and draw 50 mm of mercury to vacuum chamber.
Let biomass settle for 24 hours and measure level of lipids at surface and record.
Repeat steps # 4 through #12 for each vessel’s slurry and record.
Once each vessel’s lipids were recorded the lipids were combined together in a beaker for transesterification.
The designated supervisor was in charge of disposal. All cultures, supplies and used media will be properly sterilized in an autoclave at 125˚C (15 psi) for 20 minutes by the designated supervisor.
All work areas were properly cleaned with sanitizing spray. All safety equipment was removed and cleaned.
PROCEDURES FOR TRANSESTERIFICATION
1. All transesterification procedures were done under the direct supervision of my designated supervisor in the school lab with fume hood . All safety equipment including gloves, safety goggles, ventilation mask and apron were worn during transesterification procedures.
2. Order Methanol and Barium Hydroxide from MiniScience, Inc. 1059 Main Ave Clifton, NJ 07011. They will be stored in their original containers in a cool, dry, locked storage room until used in the experiment.
3. Review the health hazard information (MSDS) for Methanol and Barium Hydroxide with my designated supervisor. Methanol has a hazardous level of 2, flammability level of 3, and reactivity of 0. Barium Hydroxide has a hazardous level of 2, flammability level of 0, and reactivity of 1. Safety precautions include avoiding contact with eyes, skin and clothing. I must wash thoroughly after handling. I must avoid breathing the dust and keep the container closed. The Methanol and Barium Hydroxide will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, and respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the chemicals. I will wash my hands thoroughly after handling the Methanol and Barium Hydroxide.
4. Once extraction procedures were completed, the “cellulose blaster” was cleaned with distilled water under pressure.
5. CH₃OH and Ba(OH)₂ along with the captured lipid oil were placed in the cellulose blaster at ratio of 3:1 moles.
6. Assemble ultrasonic unit on top of canister ensuring unit is tightly sealed.
7. Charge canister to 147 psi (10 bars) and close gas valve. Shake canister vigorously.
8. Attach collection vessel to “cellulose blaster”.
9. Place canister upright and turn on ultrasonic unit.
10. Release pressure allowing biodiesel to evacuate canister into collection vessel. Once all biodiesel has passed through ultrasonic orifice, close gas valve.
11. Collect biodiesel from collection vessel and place in sealed beaker for
24 hours allowing settling.
10. After twenty-four hours separate the biodiesel from the sedimentation using pipette.
11. Wash with distilled water five times, using pipette to remove accumulated water from biodiesel layer.
12. Remove excess water in biodiesel by allowing a fan to aerate the biodiesel for twelve hours.
13. Record the amount of biodiesel produced.
14. The designated supervisor was in charge of disposal. All cultures, supplies and used media will be properly sterilized in an autoclave at 125˚C (15 psi) for 20 minutes by the designated supervisor.
15. All work areas were properly cleaned with sanitizing spray. All safety equipment was removed and cleaned.
The bioreactor and vessels will be constructed at home under the direct supervision of a parent.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientists, and parents.
3. Review Occupational Safety and Health Administration (OSHA) documents regarding safe practices with the use of tools (www.osha.gov) with my designated supervisor.
4. Purchase all supplies and safety equipment.
5. With adult supervision, prepare to build by putting on safety goggles, gloves, respirator mask, and lab apron in a workshop that is in a well ventilated area.
DESIGNING AND CONSTRUCTION OF THE VESSELS BY DRAWING NUMBER DESIGNING VESSELS:
1. The vessels were designed using AutoCAD. Eight separate drawings (EV-002 through EV-010) were created for each part. Three drawings (EVA-002 through EVA-004) were created to assemble the vessels.
2. I reviewed the plans with my designated supervisor for approval.
3. I sent drawings EV-002 through EV-010 to Florida Machinery to machine the parts.
4. Once the parts were machined, I cleaned and deburred all parts and assembled the vessels.
5. I utilized all safety equipment including safety goggles, gloves, respirator mask, and lab apron.
6. I put the vessels together in a well-ventilated area with my supervisor.
CONSTRUCTING THE VESSELS:
1. The vessels were constructed under the supervision of my adult supervisor and wearing all safety equipment including safety goggles and lab apron.
2. Glue the 2.54cm x 30.3cm diameter grey PVC (EV008) to each end of the 15.24cm diameter x 61cm clear PVC pipe and 19cm diameter.
3. Glue the 2.54cm x 30.3cm diameter grey PVC (EV009) to each end of the 15.24cm diameter x 61cm clear PVC pipe and 19cm diameter.
4. Glue (EV010) to (EV009).
5. Glue both .64cm x 5.1cm x 55.9cm clear PVC (EV005) inside the product created in step #1 180˚ apart. Glue 3.8cm diameter x 10.2cm PVC rod (EV003) to the 2.54cm x 13.3 diameter grey PVC (EV002).
6. Glue 3.8cm diameter x 7.6cm PVC rod (EV004) to the 2.54cm x 13.3 diameter grey PVC (EV002).
7. Place 7.6cm diameter rubber gasket to the product created in step #2 and #3.
8. Cut water fill pipe into 1.26 cm lengths to create aerator.
9. Silicone airstone to 1.26 cm fill pipe.
10. Tighten airstone assemble to EV008.
11. Bolt the product created in step #3 and step #4 to the product created in step #1 using 8 - ¼/20 x ¾ hex head bolts.
12. Teflon tape the icemaker tee valve to the drive end of the vessel.
13. Repeat steps #1 through #12 twice to create second and third vessel.
CONSTRUCTING of BIOREACTOR:
1. Designed bioreactor on AutoCAD producing drawing EV-0001. Obtained approval of drawing from my designated supervisor.
2. Purchase all supplies and safety equipment.
3. Under the direct supervision of my adult supervisor, prepare to build by putting on safety goggles, gloves, and lab apron in workshop.
4. With adult assistance, cut the particle board into two 1.3cm x 48cm x 71cm long using a hand saw.
5. With adult assistance, cut seven support brackets out of 1.27cm x 5.08 x 71cm plastic trim board using a miter saw.
6. Attach support brackets to both sides of particle board and screw together.
7. Cut 2.54cm diameter slot x 7cm long in three places.
8. Cut first slot 12.7cm, second slot 37cm, and third slot 61cm along 71cm side of bioreactor.
9. Install three on/off dimmer switches to the front panel of bioreactor and wire to each Motor and main power supply terminal block.
ASSEMBLING PHOTOAUTOTROPHIC VESSELS INTO BIOREACTOR
1. Under the direct supervision of my adult supervisor, prepare to assemble the vessels in the bioreactor by putting on safety goggles, gloves, and lab apron in workshop
2. Attach one #25 sprocket 24 tooth to drive end of each vessel (total of 3).
3. Teflon tape air hydraulic rotary union and attach to both shaft ends of each vessel (total of 3).
4. Place each vessel into a slot in the bioreactor (total of 3).
5. Install #25 sprock 12 tooth to 1.25hp gear motor.
6. Bolt #6 x 1” screw through bioreactor side wall into 1.25hp gear motor and attach #25 chain with #25 masterlink (total of 3).
7. Wire gear motors to 120 volt terminal block.
8. Wire neutral lead of gear motors to speed control pots.
9. Wire 120 volt terminal block to three prong plug.
10. Attach .32cm flex hose to air hydraulic rotary unions.
11. Repeat step #10 six times – twice for each vessel.
12. Draw vacuum in outer chamber of vessel and close off tee valve.
13. Insert filler tube from Helium supply to tee valve and open tee valve and fill to atmospheric pressure. Close tee valve.
14. Repeat #13 using Neon.
15. Label each vessel, Neon, Helium, natural.
PROCEDURES for BUILDING NUTRIENT CONTROL CHAMBER (NCC)
Under direct supervision of my adult supervisor and wearing all safety equipment including safety goggles, gloves, and lab apron, I installed the two aquarium pumps on to the pine board with 4 drywall screws.
1. With adult supervision, cut particle board into 2 pieces -1.2cm x 91cm x 61cm - using electric handsaw.
2. Cut particle board into 2 pieces -1.2cm x 66cm x 61cm - using electric handsaw.
3. Cut four pieces of pine board - 1.27cm x 5cm x 91cm.
4. Cut four pieces of pine board - 1.27cm x 5cm x 48cm.
5. Cut two acrylic sheet - .56cm x 61cm x 89cm.
6. Working in a well ventilated area and using a respirator mask, glue CPVC pipe, caps, and tee together and drill 4 holes for flex tubing insertion. Take the .32cm flex tubing and plumb from aquarium pumps to CPVC pipe assembly.
With adult assistance, drill 2 sets of holes (1.75cm) into box and glue
CPVC union and reducing bushing to box.
10. With adult assistance, drill 1 hole (2.06cm) into box and glue valve and reducing bushing to box.
11. With adult assistance, drill 1.27cm hole and install electrical cord.
Connect a clear PVC hose (.9525cm x 45.72 cm) to each port on the outside of the chamber.
Attach each hose end from the NCC to each end of the shaft on the end
caps of each vessel.
Screw four casters to bottom of NCC.
PROCEDURES FOR ASSEMBLING PHOTOAUTOTROPIC
BIOREACTOR and NUTRIENT CONTROL CHAMBER (NCC)
1. Under the direct supervision of my adult supervisor, I prepared to build by putting on safety goggles, gloves, and lab apron in workshop.
2. Place bioreactor on top of NCC and connect a 1.3cm flex hosing from the bioreactors output and input ports to the NCC output and input ports.
PROCEDURES for BUILDING THE LIPID EXTRACTOR/TRANSESTERIFICATION CHAMBER
(“CELLULOSE BLASTER”)
1. Under the direct supervision of my adult supervisor, I prepared to build by putting on safety goggles, gloves, and lab apron in workshop.
2. Screw together 3/8npt brass fitting into pressure vessel.
3. Screw together ½npt close nipple into ½npt tee fitting.
4. Screw #2 to #1.
5. Screw together 1/2npt close nipple to gas value.
6. Screw #4 to #3.
7. Screw together 200 pressure gage to reducing bushing 1/4npt to ½npt.
8. Screw #6 to #2.
9. Slide ½ high pressure rubber hose to 1/2npt x 7.6cm nipple.
10. Attach 3 hose clamps to high pressure hose.
11. Insert EV A004 drawing to #9 and attach three hose clamps to high pressure hose.
12. Attach ring terminals from ultrasonic transducer and attach with #8 screws to EV
A004 on both ends.
The following apparatuses were built for starting the algae cultures:
PROCEDURES for BUILDING PHOTOBIORECTOR
The photo-bioreactor will be constructed at home under the supervision of a parent.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientist, and parents.
Review Occupational Safety and Health Administration (OSHA) documents regarding safe practices with the use of tools (www.osha.gov) with my adult supervisor.
Designed bioreactor on AutoCAD producing drawing EV-0000. Obtained
approval of drawing from designated supervisor.
5. Purchase all supplies and safety equipment.
Under the direct supervision of my adult supervisor, prepare to build by putting on safety goggles, gloves, and lab apron in workshop.
With adult assistance, cut the particle board into four 2.54cm x 15.24cm long slot using a hand saw.
With adult assistance, cut four support brackets out of 1.27cm x 5.08 x
68cm plastic trim board using a miter saw.
9. Attach support brackets to both sides of particle board and screw together.
10. Cover side surfaces of the particle board circles with silver duct tape.
11. Cut 35.5cm diameter concrete cardboard form into 28cm length tube.
12. Attach concrete tube to support brackets in four places using drywall screws.
13. Cut reflective shield to 28cm in length and glue to the inside of the concrete tube.
PROCEDURES for BUILDING INCUBATORS
The incubators and vessels will be constructed at home under the direct supervision of a parent.
1. Read 2011-2012 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor,
qualified scientists, and parents.
3. Review Occupational Safety and Health Administration (OSHA) documents
regarding safe practices with the use of tools (www.osha.gov) with my designated
supervisor.
4. Purchase all supplies and safety equipment.
5. With adult supervision, prepare to build by putting on safety goggles, gloves,
respirator mask and lab apron in a workshop that is in a well ventilated area.
CONSTRUCTION OF THE INCUBATORS
1. Glue the 7.62cm diameter x 5.08cm white PVC pipe to the 3.81cm reducing bushing.
2. Glue the 3.81cm diameter x 2.54cm white PVC pipe to the reducing bushing and PVC cross.
Glue the 3.81cm to 2.54cm reducing bushing to the end of the PVC cross.
4. Glue 2.69cm PVC pipe to the 3.81cm bushing.
5. Glue the aquarium pumps to the PVC pipe.
6. Glue the 3.81cm x 1.27cm reducing bushing to the PVC cross.
7. File down lip on reducing bushing.
8. Glue the 22.7cm PVC pipe to the 1.27cm threaded bushing.
9. Glue the 22.7 cm PVC pipe to the 1.27cm tee.
10. Glue the 1.27cm tee to the reducing bushing.
11. Glue the 2.54cm x 1.27cm PVC pipe to tee.
12. Glue the elbow to the 2.54cm x 1.27cm PVC pipe.
13. Thread pressure gauge to end of PVC elbow for pressurized vessel only.
14. Glue 3.17cm x1.27cm PVC pipe to the tee.
15. Drill four .48cm holes on a 45˚ angle into the 3.17cm x 1.27cm PVC pipe.
16. Glue check values into each of the four holes.
17. Connect each check values to the aquarium pumps with .32cm clear hose.
18. Drill a .64cm hole into the 1.27cm cap.
19. Cut inner tube valve stem and glue it into the 1.27cm cap.
20. Glue the 1.27cm cap to the 3.17cm x 1.27cm PVC pipe.
21. Drill .95cm hole into the 7.62cm reducing bushing.
22. Teflon tape the icemaker tee valve to the 7.62cm reducing bushing.
23. Attach 25.4cm x .32cm clear hose to icemaker tee valve.
24. Teflon tape 1.27cm plug and place into 1.27 elbow and tighten to create the incubator.
25. Repeat step #14 through #25 to create the second incubator.
ASSEMBLING SPARGERS IN INCUBATORS
1. Under the supervision of my adult supervisor and wearing all safety equipment including safety goggles and lab apron, I cut water fill pipe into 45.7cm lengths.
At the bulb end, drill .32cm diameter hole through the blub at 90˚ intervals creating four holes.
Working in a well-ventilated area wearing a respirator mask, glue 10.2cm coffee stirrers into each hole using Gorilla glue.
Once glue dries, cut the coffee stirrers down to 3.18cm lengths using a ruler and scissor.
5. Slide sparger into hose bib.
Place the incubator on top of the test tube and tighten with hose clamp to create the vessel.
PROCEDURES FOR GROWING INTIAL ALGAE CULTURE
All algae will be grown and tested at the school laboratory under direct supervision of my designated supervisor.
1. Read 2012-2013 ISEF Rules and Guidelines.
2. Complete required ISEF forms and obtained signatures from designated supervisor, qualified scientist, and parents.
3. Order Spirogyra Algae Cat # 86 V 0650 from Ward’s Natural Science, P.O. Box 92912, Rochester, N.Y. 14692. The ATCC classifies Spirogyra as a BSL- 1 organism. The algae are stored in its original shipping container until separated into test tube solution.
4. Prior to testing, my supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment and algae.
All testing will be conducted in a school laboratory under the direct supervision of my designated supervisor.
While working with the algae, I will be wearing safety goggles, gloves, a protective lab coat, and a respirator mask.
7. Under the direct supervision of my designated supervisor, clean one test tube thoroughly, rinse with distilled water, and sterilize in pressure chamber.
8. Fill test tube with 1250ml of distilled water.
9. Wearing goggles, gloves, mask, and apron, add 250ml of concentrated Spirogyra biomass to the test tube from common culture.
10. Turn on each air pump to make sure sparger works. (Run for a full week before adding algae.)
11. Leave all air pumps on during the entire experiment to agitate algae.
12. Cultivate for 4 weeks until algae biomass is sufficient for transfer into vessels in photoautotrophic bioreactors.
PROCEDURES FOR GROWING and TESTING ALGAE
IN VESSELS IN PHOTOAUTOTROPHIC BIOREACTORS
All algae will be grown and tested at the school laboratory under direct supervision of my designated supervisor teacher.
1. Read 2012-2013 ISEF Rules and Guidelines.
Complete required ISEF forms and obtained signatures from designated
supervisor, qualified scientist, and parents.
Order iron powder, -100/+350 Mesh and Sodium Carbonate from MiniScience, Inc. 1059 Main Ave Clifton, NJ 07011. They will be stored in their original containers in a cool, dry, locked storage room until used in the experiment.
Purchase Rhino Power Carbon Dioxide 20oz. tank from Lowes.
Review the health hazard information (MSDS) for Iron powder with my designated supervisor. Iron powder has a hazardous level of 2, flammability level of 1, and reactivity of 1. Sodium Carbonate has a hazardous level of 3, flammability level of 1, and reactivity of 1. Safety precautions include avoiding contact with eyes, skin and clothing. I must wash thoroughly after handling. I must avoid breathing the dust and keep the container closed. The iron powder supplement and Bicarbonate will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the chemicals. I will wash my hands thoroughly after handling the Iron powder, Sodium Carbonate and algae.
Reviewed the health hazard information (MSDS) for Carbon Dioxide canister. CO₂ has a hazardous level of 1, flammability of 0 and reactivity of 0. The CO₂ will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the CO₂. I will wash my hands thoroughly after handling the CO₂.
Prior to testing, my supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment, test kit, algae and iron powder.
All testing will be conducted in a school laboratory under the direct supervision of my designated supervisor.
While working with the iron powder and algae, I will be wearing safety goggles,
gloves, a protective lab coat, and a respirator mask.
10. Split 750ml algae biomass equally into each of the three vessels.
Add 1250ml distilled water to each of the three vessels.
Add 3 grams of HCO₃ into each of the three vessels.
Attach nutrient control chamber (NCC) to each side of vessel and place in respective bioreactor.
With assistance from my supervisor, add 660ppms of CO₂ to NCC for air agitation into the vessels. Agitate for one hour.
Wearing all safety equipment and under direct supervision by my designated supervisor, stop gear motors and draw samples from each vessel to conduct cell count test utilizing hemacytometer and 400x microscope and a pH test with pH meter. I will remove a small amount of algae solution from each vessel through the tee value into a prelabeled sterilized test tube.
With assistance from my supervisor, using a sterilized eye dropper, place a small amount of the algae solution on the hemacytometer counting chamber. Dilute sample so the cells do not overlap on the Neubauer ruling grid.
Systematically count the cells in selected squares so that the total count is 100 cells or more.
Record cell count and pH reading for all three vessels.
Add in the required Na₂CO₃ to the testing sample. The amount of Na₂CO₃ added each week was determined by the pH readings. The goal was to maintain the solution at the optimal pH level of 8.2 to 8.5.
Return sample back to incubators and restart gear motors. Sterilize the hemacytometer and pH meter thoroughly. Wash hands thoroughly.
21. Each bioreactor shall be exposed to the natural light provided by the sun from
sunrise to sunset during the entire testing.
22. Under direct supervision by my designated supervisor, take weekly readings of CO₂
ppms in the NCC for 12 weeks and record data.
23. With my designated supervisor, conduct weekly cell count tests on each vessel
utilizing the hemacytometer for twelve weeks and record data.
24. With my designated supervisor, take weekly pH readings for twelve weeks and
record data for each vessel.
25. Return algae sample mixed with the required amount of NA₂CO₃ to incubators after
each cell count test and sterilize hemacytometer and test tube.
26. At the end of week twelve, terminate all CO₂ infusion to the vessels and add 1 gram of Fe₃ and 2 grams Na₂CO₃ to each vessel to promote lipid growth and flocculation.
PROCEDURES FOR FLOCCULATION
Prior to flocculation, my designated supervisor will perform a safety training session with me to ensure that I understand proper procedures for handling equipment and algae biomass.
All flocculation processes will be conducted in a school laboratory under the direct supervision of my designated supervisor. The laboratory is well-ventilated.
While working with the algae biomass, I will be wearing safety goggles, gloves, a protective lab coat, and a respirator mask.
With assistance from my supervisor, transfer all algae solution from each vessel into pre-labeled test tubes for flocculation.
5. Let algae biomass settle for 24 hours.
6. With my supervisor, record sedimentation volume using gravimetric analysis for each.
7. Wearing all safety equipment including safety goggles, gloves, respirator mask, and
lab apron, dewater each test tube to sedimentary level using a 60ml sterile syringe.
8. Place excess water in incubator samples.
9. With assistance from my supervisor, repeat step #4 through 8 for each vessel and record data, ensuring that each vessel’s algae slurry remains separate and clearly marked.
10. Wearing all safety equipment and under direct supervision by my supervisor, pour each flocculated algae slurry over the 7.6cm diameter N45 Neodymium magnet into a larger beaker attracting all the Fe² onto the magnet resulting in a clean biomass.
11. Repeat step#10 for each vessels slurry, ensuring that each vessels algae slurry
remains separate and clearly marked.
12. Use concentrated algae slurry biomass for the lipid extraction process.
PROCEDURES FOR EXTRACTING LIPIDS UTILIZING “CELLULOSE BLASTER”
All extracting procedures were done under the direct supervision of my designated supervisor in the school lab. All safety equipment including gloves, safety goggles, ventilation mask and apron were worn during extraction procedures.
With assistance from my supervisor, each vessel’s slurry was weight in labeled beakers on a gram scale. Ten percent of each vessels algae slurry was calculated and recorded.
Each slurry received 10% of their biomass weight in NaCl. The NaCl was vigorously mixed into each slurry.
Place the slurry into the “cellulose blaster’s” canister and assemble ultrasonic unit ensuring unit is tightly sealed.
Charge canister to 147 psi (10 bars) and close gas valve.
Let solution soak in osmotic bath for 1 hour.
Attach collection vessel to cellulose blaster.
Place canister upright and turn on ultrasonic unit.
Release pressure allowing solution to evacuate canister into collection vessel. Once all solution has passed through ultrasonic orifice close gas valve.
Collect biomass from collection vessel and place in vacuum chamber.
Attach hand held vacuum pump to port on lid of chamber and draw 50 mm of mercury to vacuum chamber.
Let biomass settle for 24 hours and measure level of lipids at surface and record.
Repeat steps # 4 through #12 for each vessel’s slurry and record.
Once each vessel’s lipids were recorded the lipids were combined together in a beaker for transesterification.
The designated supervisor was in charge of disposal. All cultures, supplies and used media will be properly sterilized in an autoclave at 125˚C (15 psi) for 20 minutes by the designated supervisor.
All work areas were properly cleaned with sanitizing spray. All safety equipment was removed and cleaned.
PROCEDURES FOR TRANSESTERIFICATION
1. All transesterification procedures were done under the direct supervision of my designated supervisor in the school lab with fume hood . All safety equipment including gloves, safety goggles, ventilation mask and apron were worn during transesterification procedures.
2. Order Methanol and Barium Hydroxide from MiniScience, Inc. 1059 Main Ave Clifton, NJ 07011. They will be stored in their original containers in a cool, dry, locked storage room until used in the experiment.
3. Review the health hazard information (MSDS) for Methanol and Barium Hydroxide with my designated supervisor. Methanol has a hazardous level of 2, flammability level of 3, and reactivity of 0. Barium Hydroxide has a hazardous level of 2, flammability level of 0, and reactivity of 1. Safety precautions include avoiding contact with eyes, skin and clothing. I must wash thoroughly after handling. I must avoid breathing the dust and keep the container closed. The Methanol and Barium Hydroxide will be used in a well ventilated area with an eyewash facility, sink, and fire extinguisher. I plan on wearing splash goggles, gloves, and respirator mask and lab apron throughout the experiment. My gloves will be changed each time I’m done working with the chemicals. I will wash my hands thoroughly after handling the Methanol and Barium Hydroxide.
4. Once extraction procedures were completed, the “cellulose blaster” was cleaned with distilled water under pressure.
5. CH₃OH and Ba(OH)₂ along with the captured lipid oil were placed in the cellulose blaster at ratio of 3:1 moles.
6. Assemble ultrasonic unit on top of canister ensuring unit is tightly sealed.
7. Charge canister to 147 psi (10 bars) and close gas valve. Shake canister vigorously.
8. Attach collection vessel to “cellulose blaster”.
9. Place canister upright and turn on ultrasonic unit.
10. Release pressure allowing biodiesel to evacuate canister into collection vessel. Once all biodiesel has passed through ultrasonic orifice, close gas valve.
11. Collect biodiesel from collection vessel and place in sealed beaker for
24 hours allowing settling.
10. After twenty-four hours separate the biodiesel from the sedimentation using pipette.
11. Wash with distilled water five times, using pipette to remove accumulated water from biodiesel layer.
12. Remove excess water in biodiesel by allowing a fan to aerate the biodiesel for twelve hours.
13. Record the amount of biodiesel produced.
14. The designated supervisor was in charge of disposal. All cultures, supplies and used media will be properly sterilized in an autoclave at 125˚C (15 psi) for 20 minutes by the designated supervisor.
15. All work areas were properly cleaned with sanitizing spray. All safety equipment was removed and cleaned.