Conclusion
The results from this experiment support the hypothesis that Spirogyra grown in a photoautotrophic bioreactor engineered to filter the visible light wavelengths into the red-orange light spectrum utilizing neon gas produced 14% more algae biomass than Spirogyra cultivated under the blue-green light spectrum filtered by Helium gas, and 20% more than Spirogyra cultivated in nature light. The red light emissions filtered by Neon provided the greatest penetration of light energy into the Spirogyra biomass resulting in the highest photosynthetic efficiency. Weekly pH readings showed that as the Neon vessel’s biomass grew the solution was becoming more basic than its counterparts, signifying that it was consuming more CO₂ resulting in a higher biomass production than the other vessels. Each of the three rotating double walled vessels in the photoautotrophic bioreactor were designed to expose the biomass to the same temperature, CO₂ diet, and air and mechanical agitation. The temperature range was maintained at 22-28˚C. A controlled nutrient supply chamber was built and attached to each vessel providing 12 cubic ft. of CO₂ and O₂ to the algae solution at a maximum of 660 ppms. Mechanical paddles and air agitation spargers were embedded in each vessel to ensure the algae remained suspended in the solution. An airstone was attached to the end caps of each vessel producing micro-bubbles that aided in dispersion. Light wavelengths were the only variable. Utilizing the double walled design, each vessel’s outer surround chamber was filled with Neon, Helium or the “constant,” natural air. These gases allowed visible light to be filtered into specific energy levels; the Neon vessel exposed the algae solution to red-orange spectrum; the Helium vessel exposed the solution to green-blue spectrum and the constant vessel was exposed to the entire light spectrum for the twelve week experiment. Although Helium emitted a higher energy wave than Neon, Helium released a color spectrum that was reflected by the algae’s chlorophyll; therefore, Helium’s energy waves did not penetrate the cell. Rather, Neon’s ability to emit only red-orange energy waves to Spirogyra’s cells provided the optimal photoautotrophic environment for growth.
To determine algae growth, a weekly cell count test, CO₂ airborne test, and CO₂ consumption and pH monitoring was conducted. The results are as follows:
Cell Count Test Results
Overall Hemacytometer test results showed that Spirogyra cultivated in the Neon vessel proliferated an average of 17% more than the cultures in the other testing environments over the twelve week testing period (algae remained in the vessels an additional two weeks for pH manipulation). Cultivation of the algae began in an incubator as the specimens needed an initial growing period. At the end of the four week growing period, three 250ml samples were removed from the incubator and air agitated to determine a cell count baseline for each algae sample. Each sample was placed into one of the three pre-labeled vessels and positioned into the photoautotrophic bioreactor. Weekly cell count tests were conducted utilizing a Hemacytometer by counting the individual algae cells in each Neubauer rule. At the end of the first week the algae grown in the Neon vessel showed 1071 cells per mm³ while the Helium vessel showed 1044 cells per mm³, and the natural 1062 cells per mm³. First week results indicated that the Spirogyra in the Neon vessel grew 3% more than its counterpart in the Helium vessel, and 2% more than the natural vessel. By the end of the sixth week, the Spirogyra in the Neon vessel had grown 13% more than the Helium vessel and 23% more than the natural vessel. The algae biomass in the Neon vessel had 4095 cells per mm², the Helium vessel had 4356 cells per mm³, and the natural had 3870 cells per mm³. By the end of the twelfth week testing period the biomass in the Neon vessel indicated 8280 cells per mm³ compared to the Helium vessel which had 7128 cells per mm³. The trend was clear, by varying the light energy wavelengths, Spirogyra absorbed more light energy from the red-orange light spectrum resulting in a larger biomass crop and in essence, a larger lipid yield.
CO₂ Airborne Test Results
To determine Spirogyra’s consumption rate of CO₂, a weekly airborne test was conducted in the nutrient chamber to ensure the vessels had an ample diet of CO₂. CO₂ was injected into the chamber with a baseline at 660 PPMs. CO₂ was agitated into each bioreactor through a closed loop system, wherein CO₂ was monitored for downward trends in consumption utilizing a meter. CO₂ consumption was maintained between 660– 440 PPMs. Consumption rates were recorded each week or at anytime PPMs reached the 440 PPM level. At the end of the first week, the Spirogyra biomass in all three vessels consumed 90 PPM. At the end of the sixth week, 332 PPM was consumed by the vessels. By the twelfth week, 4.5 liters of biomass had consumed 849 PPM of CO₂. Although CO₂ airborne testing confirmed that photosynthesis rates increased in direct proportion to CO₂ consumption for the first 8 weeks, after the eighth week of testing, CO₂ consumption outpaced growth rate by 2:1. This trend indicated that the optimal growing period for the Spirogyra in this experiment was eight weeks. Therefore, if utilized commercially, one could harvest six crops of biomass per year under these growing conditions.
pH Monitoring Results
To maintain an optimal growing environment, the solution’s pH was monitored on a weekly basis. The goal was to maintain the pH level between 8.2 and 8.5, the optimal level for cellular processes. Na₂CO₃ was added to the solution when it reached acidic levels which usually occurred when CO₂ was replenished. Weekly pH monitoring confirmed, Spirogyra cultivated in Neon conditions absorbed more CO₂, as the average pH level was always more basic than the other vessels’ pH levels.
Hypothesis #2
The hypothesis, that Spirogyra’s biomass can be sufficiently flocculated without the use of toxic chemicals was supported by the data collected in this experiment. A two-fold flocculation method was utilized wherein the pH of each algae solution was manipulated to a base level by adding Na₂CO₃ during harvesting and by starving the biomass of CO₂ for two weeks immediately following the twelve weeks cultivation period. By terminating the CO₂ diet, the biomass ceased reproduction and redirected its energy to lipid growth. The deficiency of CO₂ also reduced electrostatic repulsions in the biomass solution. With less repulsion, polymer chaining increased.
To flocculate, .5 grams of Fe+² was mixed into each vessel’s solution. The Fe+² bonded with the oxygen in the water and formed Fe₃O₂. Fe₃O₂ in combination with the low pH biomass solution created a polymer. This polymer caused a Coulombic interaction to occur forming matrixes on the surface of the microalgae. The matrixes caused the algae cells to form flocs resulting in a chemically-free biomass slurry that was sufficient for wet lipid extraction.
After flocculating and dewatering each vessel separately, gravimetric analysis was conducted by measuring the volume of each floc. The algae slurry cultivated in the Neon vessel measured 9.9 cm with a volume of 30.9 cm³, the Helium vessel measured 8.2 cm with a volume of 25.7 cm³, and the natural vessel measured 7.6 cm with a volume of 23.9 cm³. The Neon vessel provided 17% more slurry yield than the Helium vessel and 23% more than the natural vessel. These results confirm the hemacytometer’s findings that Spirogyra’s growth can be significantly enhanced by the energy from the red-orange wavelength filtered by Neon gas.
Furthermore, by taking into account the low set-up cost associated with ionic bonding, the fact that there is no consumption of energy or the need for toxic chemicals in the ionic process and the elimination of the time-consuming requirement of separating the biomass from filter media, ionic flocculation is a cost efficient solution for the midstream algae to biofuel process.
Hypothesis #3
The hypothesis that Spirogyra’s cell wall can be fractured using a three-fold critical cell disruption process that engages osmotic shock, homogenization and sonication to release the lipids was proven. Each vessel’s slurry was shocked with NaCl to assist in the lysing of Spirogyra’s cell wall. After the osmotic bath, the salted slurry was feed to the cellulose-blaster, an engineered aluminum tank assembly, rated for 15 bars. The slurry was forced through an ultrasonic orifice by 10 atmospheres of pressure at 1kHz frequency shearing the algae’s cell wall and membrane. After a 24 hour period, the resulting solution separated leaving a visible layer of lipids, biomass, and water. Volumetric results revealed that Spirogyra cultivated under Neon conditions had a lipid volume of 1.8cm³ as compared to 1.4cm³ for Helium conditions and 1.1cm³ for the natural conditions.
By utilizing the cellulose-blaster, the trapped lipids were able to escape in a single step wet process eliminating the high-energy costs associated with drying the biomass, chemical infusion, and solvent recovery.
Hypothesis #4
The lipids extracted from the biomass were transesterified using a homogeneous base catalysis, under ultrasonic, homogenization conditions provided by the “cellulose-blaster”. The lipids were stirred into a solution of Ba(OH)₂ and CH₄O and feed into the cellulose-blaster. The blaster was agitated for two hours to ensuring a temporary homogenous solution was achieved. The cellulose-blaster was then pressurized and the solution was forced through the ultrasonic orifice. After a twenty-four hour settling period, two layers were visible. Pending testing results, the smaller layer of green-yellow oil was assumed to be biodiesel while the whitish layer was assumed to be glycerol, pigments and Ba(OH)₂ residue.
By utilizing the cellulose-blaster for transesterification, the reaction time and static time was reduced, the amount of energy input was negligible and the chemical usage was kept to a minimum, making this process economically feasible.
Overall Trends and Concerns
This experiment proved that a four-step, energy efficient process can be developed that cultivates, harvests, extracts, and transesterifies algae into biofuel that is economically and environmentally sustainable. The double-walled photoautotrophic vessels showed that growth can be enhanced significantly by increasing light energy to the solution. But the optimal wavelength must be determined by light absorption. In this experiment, the red-orange wavelength was a better choice than the higher energy produced by the blue-green wavelength because the green colored Spirogyra reflected the blue-green wavelength but absorbed the red-orange wavelength. Aside from the growth difference, all three vessels showed an upward increase in cell count and CO₂ consumption for the first eight weeks of the experiment. After eight weeks, CO₂ consumption outpaced growth by 2:1, indicating it was time for harvesting.
The use of ionic flocculation for harvesting was acceptable, but still not at industry standards. By depleting CO₂ in solution and adding Na₂CO₃ to assist in lipid accumulation the floc was larger than the prior year’s experiment. The fact that this flocculation process is chemically free, requires minimal infrastructure cost, and requires no fossil fuel energy should be factored into the equation when determining a midstream process.
The cellulose-blaster accomplished the task of fracturing the cell wall and extracting the lipids with low energy consumption and without the use of toxic solvents. By combining the use of ultrasonic power, osmotic shock and homogenization, the pressure applied to the algae slurry was reduced while velocity was increased producing a shearing effect on the cell wall and membrane that lead to lipid release. Incorporating the cellulose-blaster in the transesterification process also aided in the overall goal of creating an environmentally friendly product that does not require more fossil fuel energy to produce than the resulting product reduces. The project’s overall goal of creating innovative processes and equipment to increase algae’s growth and synthesize its lipids into a biofuel was achieved. By eliminating costly multi-steps and harmful reactants, the production of oilgae can become both economically efficient and environmental sustainable.
To determine algae growth, a weekly cell count test, CO₂ airborne test, and CO₂ consumption and pH monitoring was conducted. The results are as follows:
Cell Count Test Results
Overall Hemacytometer test results showed that Spirogyra cultivated in the Neon vessel proliferated an average of 17% more than the cultures in the other testing environments over the twelve week testing period (algae remained in the vessels an additional two weeks for pH manipulation). Cultivation of the algae began in an incubator as the specimens needed an initial growing period. At the end of the four week growing period, three 250ml samples were removed from the incubator and air agitated to determine a cell count baseline for each algae sample. Each sample was placed into one of the three pre-labeled vessels and positioned into the photoautotrophic bioreactor. Weekly cell count tests were conducted utilizing a Hemacytometer by counting the individual algae cells in each Neubauer rule. At the end of the first week the algae grown in the Neon vessel showed 1071 cells per mm³ while the Helium vessel showed 1044 cells per mm³, and the natural 1062 cells per mm³. First week results indicated that the Spirogyra in the Neon vessel grew 3% more than its counterpart in the Helium vessel, and 2% more than the natural vessel. By the end of the sixth week, the Spirogyra in the Neon vessel had grown 13% more than the Helium vessel and 23% more than the natural vessel. The algae biomass in the Neon vessel had 4095 cells per mm², the Helium vessel had 4356 cells per mm³, and the natural had 3870 cells per mm³. By the end of the twelfth week testing period the biomass in the Neon vessel indicated 8280 cells per mm³ compared to the Helium vessel which had 7128 cells per mm³. The trend was clear, by varying the light energy wavelengths, Spirogyra absorbed more light energy from the red-orange light spectrum resulting in a larger biomass crop and in essence, a larger lipid yield.
CO₂ Airborne Test Results
To determine Spirogyra’s consumption rate of CO₂, a weekly airborne test was conducted in the nutrient chamber to ensure the vessels had an ample diet of CO₂. CO₂ was injected into the chamber with a baseline at 660 PPMs. CO₂ was agitated into each bioreactor through a closed loop system, wherein CO₂ was monitored for downward trends in consumption utilizing a meter. CO₂ consumption was maintained between 660– 440 PPMs. Consumption rates were recorded each week or at anytime PPMs reached the 440 PPM level. At the end of the first week, the Spirogyra biomass in all three vessels consumed 90 PPM. At the end of the sixth week, 332 PPM was consumed by the vessels. By the twelfth week, 4.5 liters of biomass had consumed 849 PPM of CO₂. Although CO₂ airborne testing confirmed that photosynthesis rates increased in direct proportion to CO₂ consumption for the first 8 weeks, after the eighth week of testing, CO₂ consumption outpaced growth rate by 2:1. This trend indicated that the optimal growing period for the Spirogyra in this experiment was eight weeks. Therefore, if utilized commercially, one could harvest six crops of biomass per year under these growing conditions.
pH Monitoring Results
To maintain an optimal growing environment, the solution’s pH was monitored on a weekly basis. The goal was to maintain the pH level between 8.2 and 8.5, the optimal level for cellular processes. Na₂CO₃ was added to the solution when it reached acidic levels which usually occurred when CO₂ was replenished. Weekly pH monitoring confirmed, Spirogyra cultivated in Neon conditions absorbed more CO₂, as the average pH level was always more basic than the other vessels’ pH levels.
Hypothesis #2
The hypothesis, that Spirogyra’s biomass can be sufficiently flocculated without the use of toxic chemicals was supported by the data collected in this experiment. A two-fold flocculation method was utilized wherein the pH of each algae solution was manipulated to a base level by adding Na₂CO₃ during harvesting and by starving the biomass of CO₂ for two weeks immediately following the twelve weeks cultivation period. By terminating the CO₂ diet, the biomass ceased reproduction and redirected its energy to lipid growth. The deficiency of CO₂ also reduced electrostatic repulsions in the biomass solution. With less repulsion, polymer chaining increased.
To flocculate, .5 grams of Fe+² was mixed into each vessel’s solution. The Fe+² bonded with the oxygen in the water and formed Fe₃O₂. Fe₃O₂ in combination with the low pH biomass solution created a polymer. This polymer caused a Coulombic interaction to occur forming matrixes on the surface of the microalgae. The matrixes caused the algae cells to form flocs resulting in a chemically-free biomass slurry that was sufficient for wet lipid extraction.
After flocculating and dewatering each vessel separately, gravimetric analysis was conducted by measuring the volume of each floc. The algae slurry cultivated in the Neon vessel measured 9.9 cm with a volume of 30.9 cm³, the Helium vessel measured 8.2 cm with a volume of 25.7 cm³, and the natural vessel measured 7.6 cm with a volume of 23.9 cm³. The Neon vessel provided 17% more slurry yield than the Helium vessel and 23% more than the natural vessel. These results confirm the hemacytometer’s findings that Spirogyra’s growth can be significantly enhanced by the energy from the red-orange wavelength filtered by Neon gas.
Furthermore, by taking into account the low set-up cost associated with ionic bonding, the fact that there is no consumption of energy or the need for toxic chemicals in the ionic process and the elimination of the time-consuming requirement of separating the biomass from filter media, ionic flocculation is a cost efficient solution for the midstream algae to biofuel process.
Hypothesis #3
The hypothesis that Spirogyra’s cell wall can be fractured using a three-fold critical cell disruption process that engages osmotic shock, homogenization and sonication to release the lipids was proven. Each vessel’s slurry was shocked with NaCl to assist in the lysing of Spirogyra’s cell wall. After the osmotic bath, the salted slurry was feed to the cellulose-blaster, an engineered aluminum tank assembly, rated for 15 bars. The slurry was forced through an ultrasonic orifice by 10 atmospheres of pressure at 1kHz frequency shearing the algae’s cell wall and membrane. After a 24 hour period, the resulting solution separated leaving a visible layer of lipids, biomass, and water. Volumetric results revealed that Spirogyra cultivated under Neon conditions had a lipid volume of 1.8cm³ as compared to 1.4cm³ for Helium conditions and 1.1cm³ for the natural conditions.
By utilizing the cellulose-blaster, the trapped lipids were able to escape in a single step wet process eliminating the high-energy costs associated with drying the biomass, chemical infusion, and solvent recovery.
Hypothesis #4
The lipids extracted from the biomass were transesterified using a homogeneous base catalysis, under ultrasonic, homogenization conditions provided by the “cellulose-blaster”. The lipids were stirred into a solution of Ba(OH)₂ and CH₄O and feed into the cellulose-blaster. The blaster was agitated for two hours to ensuring a temporary homogenous solution was achieved. The cellulose-blaster was then pressurized and the solution was forced through the ultrasonic orifice. After a twenty-four hour settling period, two layers were visible. Pending testing results, the smaller layer of green-yellow oil was assumed to be biodiesel while the whitish layer was assumed to be glycerol, pigments and Ba(OH)₂ residue.
By utilizing the cellulose-blaster for transesterification, the reaction time and static time was reduced, the amount of energy input was negligible and the chemical usage was kept to a minimum, making this process economically feasible.
Overall Trends and Concerns
This experiment proved that a four-step, energy efficient process can be developed that cultivates, harvests, extracts, and transesterifies algae into biofuel that is economically and environmentally sustainable. The double-walled photoautotrophic vessels showed that growth can be enhanced significantly by increasing light energy to the solution. But the optimal wavelength must be determined by light absorption. In this experiment, the red-orange wavelength was a better choice than the higher energy produced by the blue-green wavelength because the green colored Spirogyra reflected the blue-green wavelength but absorbed the red-orange wavelength. Aside from the growth difference, all three vessels showed an upward increase in cell count and CO₂ consumption for the first eight weeks of the experiment. After eight weeks, CO₂ consumption outpaced growth by 2:1, indicating it was time for harvesting.
The use of ionic flocculation for harvesting was acceptable, but still not at industry standards. By depleting CO₂ in solution and adding Na₂CO₃ to assist in lipid accumulation the floc was larger than the prior year’s experiment. The fact that this flocculation process is chemically free, requires minimal infrastructure cost, and requires no fossil fuel energy should be factored into the equation when determining a midstream process.
The cellulose-blaster accomplished the task of fracturing the cell wall and extracting the lipids with low energy consumption and without the use of toxic solvents. By combining the use of ultrasonic power, osmotic shock and homogenization, the pressure applied to the algae slurry was reduced while velocity was increased producing a shearing effect on the cell wall and membrane that lead to lipid release. Incorporating the cellulose-blaster in the transesterification process also aided in the overall goal of creating an environmentally friendly product that does not require more fossil fuel energy to produce than the resulting product reduces. The project’s overall goal of creating innovative processes and equipment to increase algae’s growth and synthesize its lipids into a biofuel was achieved. By eliminating costly multi-steps and harmful reactants, the production of oilgae can become both economically efficient and environmental sustainable.