Algae: A Truly Green Fuel to Power the World

 

Introduction/Background Research

In every pond, lake, and stream, colonies of microscopic organisms grow by the millions, thriving on sunlight and minerals. I often walk by some of the many ponds that are sprawled across my neighborhood, wondering what the encroaching, bubbling foam is made of and how it grows. The colonies are green and slimy, multiplying and intertwining their reach across the glimmering surface of the water. No, these organisms are not aliens; they are called algae. I have found that these algae produce oils that may someday replace fossil fuels and power the entire world.

Today, we look upon the alga as an organism of little importance. People think algae are slimy, dirty, and gross, and they grow in unwanted places like swimming pools and boats. However, I have found that algae show great potential as a clean, renewable alternative for our world’s energy needs, and they could become a permanent replacement for petroleum fuel in the near future. Algae have a high oil content, need little space to grow, multiply quickly, grow almost anywhere and are environmentally friendly.

Algae can produce over half their body weight in oil, and they can yield more oil per area per year than any other biomass crop (Singh). Algae can yield over 300 times more oil per acre per year than corn. They grow quickly, and in the right conditions, algae cells grow and divide exponentially, doubling every few hours. In addition, algae do not take up much space to grow.

Another benefit is that algae can grow almost anywhere in the world, including in seawater and even wastewater. Algae can purify wastewater, as they use the nutrients from the wastewater to grow. Algae can also grow on land unsuitable for other established crops, for instance arid land, land with saline soil, and drought-stricken land. This minimizes the issue of taking away land that might be used for cultivating food crops. On top of all that, algae can also reduce pollution and purify the air (Xu).

Because of the many potential benefits of algae, researchers have focused their attention in this area to tap the almost unlimited energy source of algae. However, despite the progress made over the last couple of decades, much study remains before a practical commercial solution is found. Difficulties in reproducing results in slightly different conditions, issues with scaling up experimental processes, and problems with interpreting the many factors that go into optimizing algae production, have all hindered the thrust of the technical community.

Therefore, I took up the challenge to construct my own photobioreactor design to grow and test algae under different conditions. The advantages of a closed-loop photobioreactor system are that one can keep a tight control on the growth conditions, and there is less chance of contamination. Previous research suggests that Chlorella vulgaris (El-Sheekh) and Nannochloropsis oculata (Olofsson) are some of the better algae species for producing biofuel. Therefore, I selected those two algae species for my project. One aspect of algae production that has not been thoroughly researched is its precise growing parameters under mixotrophic, autotrophic, and heterotrophic conditions.

 

Question

What is the effect of different carbon sources (glucose, glycerol, acetate) at varying concentrations (0%, 0.5%, 1.0%, 2%); different light sources (red light, blue light, a grow light); and different growth conditions (mixotrophic, autotrophic, and heterotrophic) on the biomass productivity of two algae species, Chlorella and Nannochloropsis?

 

Hypothesis

The mixotrophic condition utilizing both a grow light and the highest level of glucose tested (2%) will produce the greatest rate of algae growth for both Chlorella as well as Nannochloropsis.

 

Materials

Chemicals Mini-Photobioreactor Design
- Glycerol - four 1.25 inch PVC Threaded Plugs
- Sodium Acetate - four Air Stones
- Glucose - eight 1/8 inch X 1/4 inch MIP Hose Barb Adapter
- Superbloom Plant Fertilizer - Aquarium Silicon Air Hose (10 feet)
  - four 1/4 inch Elbow Adaptors
Microalgae Species - four Check Valves
- Nannochloropis oculata - four 1.25 inch PVC slip and Thread Adapter
- Chlorella vulgaris - four 1.5 inch PVC "repair" Couplings
  - four 2 inch PVC "repair" Couplings
Measurement of Growth (Instruments/Techniques) - four T-12 36 inch Fluorescent Tube Protectors
-120 Glass Vials (50ml) - 4-Way Aquarium Gang Air Valve
-Digital Lux Meter (model LX1010BS) - Air Pump
-Hanna pH Digital Meter (HI 9813-5) - Dynaflex 230 Sealant
- Motic BA350 Bi-Focal Light Microscope - Digital timers
- Algae Growth Color Scale Chart  
- Microalgae Density Stick (Secchi Disk)  
   
Light Sources  
- Red Light-Emitting Diode (LED) (5m, SMD 5050, 300 LEDs)  
- Blue Light-Emitting Diode (LED) (5m, SMD 5050, 300 LEDs)  
- Grow Light-Emitting Diode (LED) (5m,SMD 5050, 300 LEDs)  
 

Procedures

The initial experimental setup

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First, I constructed the vial tester experiment in my room. The vials were divided into categories and placed onto one of three shelves. The vials were lined up in one row to get even lighting. The top shelf had a grow light, the middle shelf had a red light, and the bottom shelf had a blue light. The control experiment called for no light, so those were conducted inside my closet, where there was no light.

The control group

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Next, I inoculated the vials necessary to carry out the experiment. Ten jars were filled with 500 ml of distilled water, and 3/10 grams of Superbloom were added to each jar as food. An appropriate amount (2.5 g, 5 g, 10 g) of acetate, glycerol or glucose was added to each jar. The last jar had no carbon sources added. Then 22.4 ml of incubated algae (at 44.8 million cells/ml) was added to each jar, to end up with a final concentration of 2 million cells/ml of algae. From the appropriate stock jar, 30 ml of the stock solution was added to each 50 ml glass vial.

Next, I built my own four-tube photobioreactor (PBR) setup to test algae on a larger scale. The PBRs were inoculated initially by filling two clean milk jugs each with two liters of distilled water and 1.2 grams of Superbloom. Forty grams of acetate was poured into one jug, with nothing added to the second. Each jug was then mixed with 769 ml of Nannochloropsis (containing 8.8 million cells/ml), thereby diluting the concentration to 1.0 million cells/ml. One liter was then added from the appropriate jug to each of the 4 PBRs. The algae growth chart, a Secchi disk, and a lux meter were used separately to measure the growth of the algae over time. All light sources were hooked up to a digital timer that was on for 12 hours and off for 12 hours.

Andy inoculating vials (left): The four-tube photobioreactor (right)

Andy inoculating vials (left): The four-tube photobioreactor (right)



 

Results and Discussion

 

1) Vial Experiment

The first experiment looked at two different algae species, Nannochloropsis oculata and Chlorella vulgaris, using 50-ml vials, and was run as a screening experiment to see which variables were most important for a second, larger, scaled experiment. The variables examined were the three different carbon sources and for each carbon source, four different concentrations. Furthermore, different wavelengths of light were also examined under each condition listed above. Three replicates were done to gauge the reliability of the results. This experiment ended up with 120 total vials measured on a daily basis, tracking the growth of algae over time.

Andy examines the 120 vials (left): The vial experiment (right)

Andy examines the 120 vials (left): The vial experiment (right)


A sample of what was seen with regard to the variation in color from the vials is shown in the picture.

Variation in color can be seen in the vials.


The tables show the highest levels of algae concentration measured for a given condition over the length of the experiment. The optimal carbon source for Nannochloropsis was found to be acetate, compared to glycerol and glucose. The optimal concentration of acetate for Nannochloropsis was 2%, and the best light source was the grow light. A grow light contains light at multiple wavelengths, which made it ideal for adsorption by the chlorophyll in the algae and helped the algae grow.

 

Algae Growth Charts for Nannochloropsis

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Maximum Growth for Given Condition for Nannochloropsis

nanno growth table summary

 

Algae Growth charts for Chlorella vulgaris

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Maximum Growth for Given Condition for Chlorella vulgaris

chlorella growth table summary

Similarly, Chlorella grew best with acetate. However, Chlorella seemed to grow better with acetate at a concentration of 1%. The Chlorella died off with 2% acetate, suggesting that the concentration was too high, acting probably as a poison. This revealed that the optimal level of concentration of carbon sources can be different when comparing one alga species to another.

 

2) Mini-PBR Experiment

The second experiment looked at Nannochloropsis using larger, home-built mini-PBRs with acetate at 2%, based on results from the experiment with the vials. There were four mini-PBRs, with the first running under mixotrophic conditions (2% acetate, grow light), the second under heterotrophic conditions (2% acetate, no light), the third under autotrophic conditions (no acetate, grow light), and the fourth under control conditions (no acetate, no light).

Two different attempts were conducted before successful growths were seen. The first failure was linked to the fluorescent light bulbs initially used as light sources. The algae were not growing, and it was noticed that there was a lot of heat coming from the light sources. A digital thermometer was installed to monitor the temperature, and it was found that high temperatures (32ºC and above) were killing the algae. Alternate sources of light were investigated, with LED lights being selected due to their low release of heat.

Andy uses a Secchi disk to measure the growth of Nannochloropsis.

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The second attempt failed due to a decline in pH levels in the mini-PBRs. The excess agitation caused by bubbles from the air pump led to production of carbonic acid, which resulted in a decline of pH levels. This problem was fixed by taking measurements with a pH meter daily and correcting the pH with baking soda. The bubbling was also kept to a minimum. With these changes, the algae flourished and grew well.

The growth of the Nannochloropsis was measured using the Secchi disk method for the four different growth conditions (mixotrophic, heterotrophic, autotrophic, and the control). As shown in the graphs below, a mixotrophic condition of 1% acetate under a grow light in PBR1 was found to be the best for growing Nannochloropsis.

 

Growth Chart for the Mini-PBR Experiments using the Secchi Disk

mini pbr cell population

 

 

Comparison of Growth conditions using Secchi Disk Method


The growth of the Nannochloropsis as determined by a different assessment technique—the algae color chart method—is depicted in the figures below. The mixotrophic condition of 1% acetate under a grow light in PBR1 (utilizing the algae color chart) was found to be the best, similar to the results from the Secchi disk method.

 

Growth Chart for Mini-PBR Experiment using Color Scale

mini pbr color scale

 

Comparison of Growth Conditions Using Color Scale Method

mini pbr color scale comparison

 

Andy taking readings with the lux meter

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The novel idea of using the lux meter as an assessment method was then tested to measure algae growth. The photo detector was placed close up behind the PBR as light was shining through the algae, and measurements were made. The numbers on the lux meter dropped when the algae became denser because the light penetrated less. The mixotrophic condition containing a higher density of algae led to lower readings of percentage transmittance (see graph below). After a certain period of time, the algae in the photobioreactor tube became too concentrated for much light to pass through. From then on, readings from the lux meter remained fairly constant, as greater growth in algae could not be captured by this technique.

 

 

 

Growth Chart for Mini-PBR Experiment using the LUX Meter

mini pbr lux

The measurements of algae growth using three different techniques then were compared in the graph below. The data was normalized to 1, and relative changes in the measurement for a particular technique were calculated compared to its maximum. The comparisons were very good, and this would suggest that the use of a color scale could be an easy, quick, nonintrusive way to measure algae growth.

Although the experiments were planned to minimize experimental errors, several sources of error were identified. The air pump in the mini-PBR setup was highly sensitive, with valves that were sometimes hard to control, resulting in minor uneven bubbling between the tubes. Purchase of more expensive valves might alleviate this problem. Two of the measurement techniques (the color chart and the Secchi disk) have the potential for subjectivity, as they depend on human perceptions of color, which can vary from person to person. However, triplicates were done on every point to minimize that error.

Future studies could look at comparing other measurement techniques, such as the use of a haemocytometer with a microscope, measurement of the algae’s dry weight, or the use of spectrophotometer with the techniques used in these experiments. This would further test the validity of the results from this science project. Additional experiments to explore, using the techniques defined in this project, would be the effect of other carbon sources than the ones used here on the growth of algae, or to look at different algae species to investigate potential synergies when they are combined.

 

Comparison between Different Algae Measurement Techniques

mini pbr relativer ration different techniques

Conclusion

In conclusion, my hypothesis was partially supported. As expected, algae growth under mixotrophic conditions exceeded the growth under heterotrophic as well as autotrophic conditions for both Nannochloropsis and Chlorella. The use of both routes, involving photosynthesis under light as well as the carbon consumption process, was seen to increase algae growth, with less biomass loss during the dark phase.

The results revealed that sodium acetate was the best carbon source for growth in both species of algae. It was interesting, however, to find that the optimal levels of sodium acetate for growth were different between the algae species (2% for Nannochloropsis and 1% for Chlorella). The higher concentration of acetate for Chlorella probably acted like a poison, while the Nannochloropsis was unaffected by this slight change.

Multiple techniques—the Secchi disk, light transmittance by using lux meter, an algae color growth chart—were all successfully used to track the growth of the algae. Results from the mini-PBR experiment supported the conclusions seen from the vial experiment. These multiple tracking techniques could be an easy, quick, nonintrusive way to measure algae growth by others in the future.

This study demonstrates that examining growth conditions is critical in enhancing algae growth, and that researchers need to continue their work on the potential viability of algae as an alternate energy source. Algae biofuels are quickly and efficiently produced, environmentally friendly, and this alternative green “pond scum” might be the perfect solution to what the human race needs in the future. One can envision the day when the roads might be filled with algae-fueled automobiles and factories run by algae-produced oil, and the environment and the world will be safer because of it.

 

Acknowledgements

Many thanks to the following people for their help and time:

  • Mr. Ron Riley, director of the National Algae Association, for the Nannochloropsis algae culture, the Secchi disk instrument, and advice on how to get started with growing algae.
  • Ms. Michelle Coleman, researcher and head of the Algae Lab at Huntsman Company, and Dr. George Smith, principal fellow at Huntsman Company, who gave not only their time but also assisted in mentoring and advising me on the different aspects of algae research. Their invaluable expertise and willingness to help have been inspiring. Also, the Chlorella vulgaris algae culture was provided by Ms. Coleman and Dr. Smith.
 

Bibliography

Abreu, Ana, Bruno Fernandes, Antonio Vicente, Jose Teixeira, and Giuliano Dragone. “Mixotrophic cultivation of Chlorella vulgaris using industrial dairy waste as organic carbon source.” Bioresource Technology 118 (2012): 61-66. Print. 

El Nabris, Kamal. “Development of Cheap and Simple Culture Medium for the Microalgae Nannochloropsis sp. Based on Agricultural-Grade Fertilizers Available in the Local Market of Gaza Strip.” Journal of Al Azhar University-Gaza 14 (2012): 61-76. Print.

El-Sheekh, Mostafa, Mohamed Bedaiwy, Mohamed Osman, and Mona Ismail. “Mixotrophic and heterotrophic growth of some microalgae using extract of fungal-treated wheat bran.” International Journal of Recycling of Organic Waste in Agriculture 1 (2012): 1-9. Print. 

Olofsson, Martin, et al. “Seasonal Variation of Lipids and Fatty Acids of the Microalgae Nannochloropsis oculata Grown in Outdoor Large-Scale Photobioreactors.” Energies 5 (2012): 1577-1592. Print.

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Singh, Jasvinder, and Sai Gu. “Commercialization potential of microalgae for biofuels production.” Renewable and Sustainable Energy Reviews 14 (2010): 2596-2610. Print.

Xu, Jingyang, Wei Du, Xuebing Zhao, Guoling Zhang, and Dehua Liu. “Microbial oil production from various carbon sources and its use for biodiesel preparation.” Biofuels, Bioproducts & Biorefining 7 (2013): 65-77. Print.