Introduction: DIY Proof-of-concept Spirulina Photobioreactor to Reduce CO2 Emissions
Global warming is progressing at an alarming rate and has become a critical issue for the environment. To reduce emissions carbon dioxide sequestration (removal of CO2 from the atmosphere) technology has been considered. Using algae as carbon sinks is simple, sustainable, and environmentally friendly. Arthrospira platensis (Spirulina) has a high capability for CO2 fixation while being a very useful crop itself. It is a superfood and can be used as a source for biodiesel, bioplastics, and fertilizers. Building a photobioreactor (PBR), and eventually scaling it up, can optimize the growth of Spirulina to maximize the amount of CO2 removed from the atmosphere as well as the amount of useful biomass to be harvested.
Step 1: Reactor Container and Components
The reactor container chosen for this project was low density polyetheylene (LDPE) tubing. Multiple bags can be used in parallel to grow large amounts of algae in a short time. Furthermore, plastic bags are cheap, easy to hang indoors (to save space), and LDPE is known to be permeable to CO2. A secondary container is highly encouraged, especially when growing indoors, as the LDPE bags have a risk of bursting/leaking if left to sit for long periods of time (months at a time). In order to facilitate optimal conditions for Spirulina you will need to maintain temperature, CO2 and O2 levels (via aerator), and lighting. To harvest Spirulina you will need a filter with a mesh size of at most 35 microns such as a coffee filter which typically has a mesh size of 20 microns.
Bon Tool 11-407 Utility Tub - 26" X 20" X 6 1/2" (secondary container)
For Data Collection:
Temperature Sensor (+ Terminal Adapter needed for connecting temperature sensor to arduino)
Additionally, you will need a starter Spirulina culture kit from Algae Research and Supply and bottled water. The biomass of Spirulina harvested at the end of the project can be increased by increasing the culture volume gradually during the culture process. Hence, purchase as much water, nutrient media, and culture salts as you will need for your desired final harvested mass of Spirulina. Optimistically, a yield of 0.16g to 0.32g of Spirulina per litre of culture volume can be expected.
Step 2: Finding a Space to Hang the Reactor
I was able to use this metal desk frame with a glass top to hold my lamp and hang my plastic tubing from. A similar setup can be done in any home space with the criteria below.
Reactor setup space requirements:
An indoor space where noise from aeration and lighting will not affect one's well being.
A maximum of 3.5ft away from a power outlet in order for the reactor components to access power.
A shelf or platform to place the lamp above where the plastic tubing will hang. The fluorescent lamp linked in step 1 includes a metal chain to hang the lamp as well if there are hooks or holds that are sturdy enough for the weight of the lamp.
[Optional] A maximum of 3ft away from a surface to place a computer and arduino for recording data (such as turbidity and temperature) automatically.
Step 3: Growing Your Culture
To start growing your culture follow the directions in the booklet that comes with the 1.7 mL spirulina culture kit ordered from Algae Supply and Research. The culture should be placed in dim light for the first two days than in good light (from a fluorsecent bulb or even in indirect sunlight on a windowsill) and kept in the range of 22-28 degrees celsius. Ensure to shake the flask at least twice daily.
Once the culture has grown quite dense in the flask (similar to pictures above of the 3 bottles of culture at day 45) consider moving the culture to another container such as a plastic water bottle. If you need to produce more culture media (dissolved salts and nutrients for the algae) follow the recipe below and maintain the same ratio of salts, nutrients, and water:
For 500 mL (common size for plastic water bottles) of water:
Add 7.93 g of salts
Add 1.32 mL of nutrients
Shake until all solids are dissovled
To increase culture volume from the original flask move the culture to a larger container and add more culture media a ratio of 1:1 in terms of algae culture to culture media. This can be repeated every 3 days, but observe the color and density of the algae to ensure that it is dense enough for a volume increase. Continue to shake the containers with the increased culture volume at least twice a day.
To be more precise in determining when to double the culture volume one can purchase a secchi stick (a manual device to determine turbidity, a formal measure for determing the density of algae in the culture). The secchi disk should not read above 5cm before increasing culture volume.
If the culture does not look healthy (yellowish in color and clumping together at the bottom of the flask) it is highly reccomended that you separate the mixture into 3 different containers and place them in different conditions. This is most commonly due to lack of nitrogen or an excess of light.
In my case, I noticed this symptom on day 14 and separated the culture. I placed a container in dim light, one near the windowsill (to receive more air) and indirect sunlight, and the third one under a brighter fluorescent bulb. Since transferring the culture into the different flasks I had no more problems with growing the algae culture and had about 1.5L of dense culture by day 45 after multiple culture dilutions and volume increases.
Step 4: Mounting Heating and Aeration Components to Reactor
Once your setup space is determined and fits the requirements outlined in step 2 you can begin setting up your reactor.
Method: [for a volume of 2L]
First, place the secondary container under where the bag will go. Next, cut a 3.5ft section of LDPE tubing. The excess length will be used for tying the bag around a hook or around a bar to be hung over the secondary container. After the bag is in place and securely tied cut an opening at a height well above the calculated length of tubing required for the desired target volume (1.05ft for 2L).Then, through those openings, carefully place the heater and the water stones (attatched to the airlines connected to air pump) at the bottom of the bag.
Notes:
You will need to determine what length to cut your tubing. This will depend on how much biomass you're planning to cultivate. 2L is a manageable amount of culture to aim for at first without taking up too much space.
To show the components within the reactor bag clearly I demonstrate the setup with water only.
Step 5: Transfer Culture and Add Culture Medium to Reactor
Don't start this step until you have grown enough dense algae culture to transfer about 1L into the reactor bag.
Method:
For 2L starting volume in the reactor:
Add the 1L of dense algae culture to the reactor bag.
Prepare 1L of culture medium (using the recipe in step 3) and add it to the reactor bag.
Notes:
Transferring the medium and the culture to the bag should be relatively easy to do if you've been using plastic bottles to hold your culture. If you're using a container that does not easily allow for accurate pouring a funnel may be of aid.
Step 6: Mark Water Level, Clip Bag, and Maintain Culture
Method:
Clip your bag at the top to minimize the area exposed to the air as to capture more vapor so it can condense and drip back into the reactor. It is also important to mark the water level (with a marker or tape) at the start of operating the reactor to refill the reactor with culture media when needed. Now that the reactor is assembled switch on all the components and set the heater temperature to 30C. Monitor the health of the algae culture daily by judging its color (deep green is what you're aiming for). Continue to add culture media when the water level drops as needed.
Notes:
When using a water heater evaporation is accelerated, so be mindful of maintaining the culture volume. A 10 hour test with water only and a heater setpoint temperature of 30C in the reactor showed no significant difference in water level. However, for the reactor running at 35C (the upper limit at which temperature is optimal for spirulina) I noticed a signifiant drop in the water level. Taking readings every 10 hours I recorded a drop in water level at a rate of about 1cm every ten hours and 3.2cm over the course of the total 40 hours in which I monitored the reactor system.Thus, it is important to mitigate water loss as it will need to be replaced to maintain the healthy growth of spirulina.
Step 7: Harvest Your Spirulina
Method:
To harvest your spirulina use a coffee filter to capture the algae while pouring your culture over it. To keep the filter in place use a jar or pitcher and an elastic band (I used a hair tie band) as shown in the picture. Be patient while pouring as it can overflow easily and spirulina yield will be lost. Afterwards, let the spirulina dry on the coffee filter.
After accounting for the weight of the coffee filter I measured a dry mass yield of 0.52g of spirulina.
Notes:
Alternatively, you can purchase an algae harvesting screen from Algae Research and Supply. The harvesting screen will be more efficient at collecting algae and mitigating biomass loss during the transfer of the culture. However, it is significantly more expensive than a coffee filter.
Also, it may be very difficult to harvest small amounts of algae as you must scrape it off the filter. I reccommend only harvesting your algae once a visibly large amount has been collected at the surface of the culture or your culture looks very dense.
Step 8: Dispose of Your Spirulina
Method:
You can dispose your spirulina easily by treating it with 10% bleach solution. Add one concentrated bleach tablet to 1 quart of water in a bucket (a small trash bin was used here). Wait until the tablet dissolves. Pour your spirulina into the bucket and leave for 24 hours. After the spirulina is treated with bleach you can pour the mixture down the drain and continue to pour water until the odor of bleach is no longer present.
Notes:
Spirulina can be very harmful to the environment. Spirulina is a type of cyanobacteria which are known to release 100 times more hydrocarbons than petroleum scources. Thus, it is crucial that if you are not harvesting your algae you must dispose of it in an enviornmentally concious manner.
Step 9: Waterproofing Your Turbidity Sensor for Data Collection
Method:
1) Use a hot glue gun to seal any cracks around the sensor where water could seep in.
2) Once a full seal has been made let the glue dry for a minute.
3) Use duct tape and plastic tubing to further cover the top half of the sensor
4) Use the same LDPE tubing to cover the exposed wire coming from the top of the sensor
a) Cut a section of tubing approximately 2.5 in in diameter and 10 in in length.
b) One side of the section will be open and unsealed - seal it lengthwise with the impulse sealer.
c) Insert the glued/taped turbidity sensor through the tubing.
d) Use rubber bands to keep the top and bottom of the wiring tightly covered in the LDPE plastic wrap.
e) Use duct tape to seal the plastic wrap onto the turbidity sensor.
5) For good measure, double check the sensor and seal any visible cracks with hot glue.
Notes:
Before purchasing from DFRobot I had not read in the reviews that the top of the sensor was not waterproof. As a result the inside of the tip of the sensor became waterlogged relatively quickly. The readings went from varying (due to bubbles from the aerator getting in the way) to constant around 1.57V (due to the sensor not being able to measure anything externally). So, I would reccomend ordering another sensor as a backup.
In case your sensor does get waterlogged I reccomend placing it in an airtight jar of rice for a few days to to dry it out.
Step 10: Data Collection and Optimal Maintenance
Method:
Once your culture is setup in the reactor and the heating, aeration,
and lighting components are working. You can start mounting the automatic measurement components. Mount the temperature sensor and ensure it is not too close to the water heater in order to measure an accurate reading of the bulk culture. Next, mount the turbditiy sensor at the top of the bag just as to let the bottom tip of the sensor sit in the water. To adjust the depth/position at which the sensors sit in the water use the white plastic clips to attach there connecting wires to the reactor bag itself.
To use the automatic sensors connect them as directed by their documentation on dfrobot.com and follow the example code available. To log and save data collected with arduino one can use software such as CoolTerm.
Notes:
The reactor should now look similar to the setup shown in the pictures above.
Ensure the turbidity sensor is distanced from other components that could interfere with its readings. For example if there is a wire between the two plastic tips the sensor could mistake it for a increased turbidity.
To improve from the previous steps it is important to maintain spirulina in its optimal conditions in regards to pH, temperature, CO2 levels, and O2 levels. Although one can visually check for spirulina health by checking the culture color (deep green and not yellowish indicate healthy culture) it may be more precise to use microscopy. With a microscope one can check the number of spirals in a spirulina filament. Generally, a filament with four or more spirals is considered healthy. Using this metric you may be able to fine tune your reactor conditions to produce higher yields of spirulina biomass.
In this project however a microscope was not used. Here, the only metric used in evaluating spirulina was turbidity. This can be done using a turbidity sensor or a secchi stick.
While evaluating growth collect data on other stimuli such as temperature, pH, CO2, and O2 levels to see how growth changes when each of those factors fluctuate. To do so you will need to choose whether you will monitor these stimuli automatically or manually and how frequent you should sample for them. For this project I was able to use an arduino to program a turbidity sensor and temperature sensor to automatically record data for a period of days.
Step 11: Planning Experiments
Evaluation Metric: The metric used for experiments can be whatever you choose, but here I used turbidity readings as a metric for evaluating the growth of spirulina.
Independent Variable: This is also up to the reader to choose. Some easy suggestions would be lighting schedule, lighting wavelength (color), aeration schedule, and temperature. Here, I chose to vary temperature in two sets of experiments. To put the optimal range of temperature to the test for spirlulina I planned to maintain the reactor at 30C and 35C for 72 hours in two separate experiments.
Other Stimuli to Control: During these experiements it's important to maintain unchanged control over any other stimuli such as light, pH, CO2, and O2.
Duration: 72 hours was the reccomended time to double culture volume during the early stages of culture growth, hence, I thought that would have been a good timeframe to see a noticeable difference in turbidity.
Step 12: Experimental Results and Interpretation
Experiment 1: 30C for 72 hours
The experiment ran for the full 72 hours with some interruptions in heating and aeration. The results indicate a trend between turbidity and temperature when looking at the voltage results. However, after converting turbidity as a sensor reading in terms of voltage to a value in NTU using the formula given in the DFrobot wiki it is clear the turbidity trend is not due to temperature. When the aeration unit is switched off, which I did during the night to avoid disruptions to sleep, the algae gradually settles to the bottom of the reactor leaving most of the culture surprisingly transparent leading to very low values of turbidity.
To address this I removed the data points during which the aerator was switched off. The line of best fit changed from a positive gradient to a negative one, however the R squared value was only 0.0007.
Experiment 2: 35C for 72 hours
The experiement did not run for the full 72 hours. The culture appeared unhealthy (had turned slightly yellow - see image above) after about 30 hours. The color of the culture did not improve and turbidity remained constant so the experiment was stopped early at 39.4 hours in. I also accounted for the lack of aeration during the nights and found the gradient of the best fit line to be negative.
Interpretation:
Based on the culture health in the second experiment it was clear that 35C was too much heat for the algae. Nonetheless, the gradient of the best fit line for turbidity was less negative than the gradient of the best fit line in the first experiment. Although both regressions revealed a downwards trend in growth (by their negative slopes) I am no longer certain that turbidity alone is a good metric when conditions are not optimal, which is expected outside of a laboratory setting. This is due to the effect of dead spirulina cells adding to overall turbidity when they are not an indication for growth.
