Enhancing the growth of Chlorella vulgaris using indoor bioreactors

supplemented with CO2 gas.

Students: Barnes, Joseph, A.Quiñones, Maria

Advisor: Torres, Hirohito, Phd, PE

Physics and Chemistry Department

Industrial Chemical Processes Technology

(Accredited by ABET)

Eleventh Undergraduate Research Forum

December 12, 2014

UPR - Arecibo

Introduction: Chlorella vulgaris, a multipurpose microalgae.

C. vulgaris is a unicellular green microaglae, a photoautotroph which generates sugars, lipids and proteins via oxygenic photosynthesis.

It is one of the most investigated microalgae because of its multiple uses and multifaceted properties, including its high concentration of lipids that can be transformed into different types of biofuels.

Multipurpose Microalgae:Industrial Uses

Included among its potential industrial uses are the following:– Generation of biodiesel (C. vulgaris has a high

lipid content).– The industrial production of ethanol from corn and

switchgrass plant (it posses cellulose degrading enzymes).

– As a water bioremediation agent.– For the production of pigments (it holds a high

content of chlorophyll).

Multipurpose Microalgae:Industrial Uses

Multipurpose Microalgae:Medicinal Uses

Currently C. vulgaris is also being studied for medicinal purposes, including: – As a chelating agent for its ability to remove

heavy metals in the human body.– As an active ingredient (Dermachlorella) for skin

cream, for restoring the elasticity of the skin (e.g. remove wrinkles and stretch marks).

– It is used for reducing problems caused by exposure to harmful radiation, such as UV radiation.

Multipurpose Microalgae:Medicinal Uses

Biochemical Composition of C. vulgaris

C. vulgaris has a high lipid content, up to 40% or more.

Lipids can be extracted and converted into biodiesel via transesterification.

Lipid vacuoles or droplets within the cell, visualized by TEM, transmission electron microscope.

Benefits of Producing Biodiesel Using Microalgae like C. vulgaris

Relatively rapid growth rate in shallow bodies of water or in photobioreactors, without the use of excessive square-miles of surface terrain as in the case of other oil-producing crops (e.g. coconut trees, corn, soybeans, etc.)

Sequestering vast quantities of carbon-dioxide. Growing microalgae can be a carbon-neutral process, that is, the carbon dioxide emitted by the burning of biodiesel is reabsorbed by the microalgae.

Ability to utilize heterotrophic metabolism in order to absorb sugars and other organic-carbon substances, thus it proves a viable means for removing industrial waste and unwanted byproducts.

Long-Term Objectives

To grow C. vulgaris microalgae in a cost efficient manner, using the most economic resources available.

To reach a growth rate and biomass production

(> 1 g/L) sufficient for lipid extraction. To extract the lipid content and convert it to

biodiesel. Devise a means to couple CO2 sequestration with

biodiesel production.

Short-Term Objectives

Establish growth conditions for optimal production of microalgae biomass.

Enhance microalgae production by supplementing growth media with CO2 gas.

Establish optimal input flow for CO2 gas. Generate concentrations of microalgae

biomass which exceed 1 g/L.

Materials:Equipment set-up

1-liter reactor bottles. Growth media (Commercial grade fertilizer). Aquarium air pumps (average flow rate: ~ 660 ml/min). Growth lamp (luminous flux = 660 Lumens) with high-

intensity light emissions at red and blue wavelengths. CO2 gas tank with regulator valves. Gang valves. Hemocytometer. Light microscope.

Materials: Equipment set-up

Materials:Equipment set-up

Procedures Implemented and Results Recorded

Hypothesis:

Feeding C. vulgaris, an unicellular autotroph, with CO2 gas at a high flow rate will increase its cell concentration.

Method 1

The control group and experimental group were prepared in duplicate, using distilled water supplemented with 0.75 g/L of 20-20-20 commercial grade fertilizer inoculated with a fresh culture of C. vulgaris.

The reactors bottles were placed in an Orbital Shaker set to stir at 125 rpm.

The growth lamp was regulated to a 12hr/12hr light/dark cycle by the timer.

The experimental group was exposed to CO2 gas at a high flow rate (~ 620 ml/min) for a an exposure rate of 40 min every 1 hour of light.

Results Following Method 1:Final cell concentrations (106 cells/ml)

After 8 days of growth, we measured cell concentrations via a direct cell count using the hemocytometer and the light microscope.

As seen on the graph, the control group flourished in comparison to the experimental group.

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Method 1: 8 days

Control

Experimental

Method 2

We repeated the procedure used in the anterior experiment, however, we reduced the rate of exposure to CO2 gas from 40 min/hr-light to 20 min/2hr-light (8 hr/day to 2 hr/day).

At the end of 8 days, we measured the final cell concentrations via direct cell count using the hemocytometer.

Results Following Method 2, Combined with Those of Method 1:Final cell concentrations (106 cells/ml)

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Method 1:8 days

Method 2:8 days

Control Experimental

Method 3

We repeated the procedure used in the 1st attempt, however, both the control and the experimental groups were aerated.

A gas line carry CO2 gas was connected to the airline which aerated the experimental group. The air flow was mixed with CO2 gas (~500 ml/min).

The exposure rate to the air mixed with CO2 gas was limited to 30 min/2hr-light (3 hr/day).

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Method 1: 8days

Method 2: 8days

Method 3: 13days

Control

Experimental

Results Following Method 3, Combined with Those from Methods 1 and 2:Final cell concentrations (106 cells/ml)

Immediate Observations Regarding Direct CO2 Supplementation

Direct injection of CO2 at high flow rates inhibits cell growth and reproduction of C. vulgaris.

Addition of CO2 gas at a more regulated rate (diluted with a strong airflow) sustains growth.

Method 4:Determining Optimal CO2 Dilution Rates

We prepared 6 bioreactors, arranged into two groups, control (2 reactors) and experimental (4 reactors). Both groups were well aerated. All bioreactors had 0.75 g/L of 20-20-20 fertilizer.

The airflow for each bioreactor composing the experimental group was mixed with CO2 gas at a flow rate distinct from the rest.

Dilution rate determined by measuring the fraction of total gas volume occupied by CO2 gas:

– CO2 gas flow/ (CO2 gas flow + airflow) x 100%

Results Following Method 4:Final cell concentrations (106 cells/ml)

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1 2 1 2 3 4

Control Experimental

Exp.Units

CO2 gas flow rate

CO2 % of total volume

1 403 ml//min

38%

2 93.6 ml/min

12%

3 36.7 ml/min

5.2%

4 9.36 ml/min

1.4%Cell counts were performed following 16 days of growth

Immediate Observations Following Method 4

Higher concentrations of C. vulgaris were attained at higher CO2 dilution rates, with optimal growth where CO2 is at 5% of total volume.

Unable to measure growth rates.

Final cell concentrations are determined by carry capacity.

Highest dried biomass reached was 0.43 g/L.

Other Observations:Observed Growth Curve

During its observed life cycle, C. vulgaris undergoes certain patterns of development.– Inoculum in fresh growth medium undergoes a

growth phase (log phase), generating a dark green medium with low transmittance.

– Following a given period of time, cells begin to clump or agglomerate, eventually forming flocculants which tend to precipate on available solid surfaces. The medium loses its green color and remains clear with a high transmittance.

Growth Curve in a Static System

Other Observations: Why Excessive CO2 May Inhibit Growth

Dissolving CO2 in water will generate carbonic acid (H2CO3), lowering the pH and increasing the acidity of the solution.

The pH may drop below the acceptable range for sustainable growth.

CO2 (aq) + H2O H2CO3 (aq)

Conclusions

Direct infusion of CO2 gas at a high flow rate inhibits growth.

Diluting the CO2 gas in airflow at regulated levels will enhance the growth C. vulgaris.

The optimal level of CO2 in the airflow is around 5%. Biomass readings fell short of the 1g/L mark, however

there were higher than earlier attempts: (~30 days) Spring 0.30 g/L; (28 days) Summer 0.39 g/L;

(19 days) Fall 0.43 g/L. Feeding CO2 is more complicated than earlier assumed.

References

Cheirsilp, B., Salwa, T., 2012. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light intensity, glucose concentration and fed-batch cultivation. Bioresource Technology 110, 510-516

Debjani, M., van Leeuwen, J.H., Lamsal, B., 2012 Heterotrophic/mixotrophic cultivation of oleaginous Chlorella vulgaris on industrial co-products. Algal Research 1, 40-48.

Leesing, R., Kookkhunthod, S., 2011. Heterotrophic growth of Chlorella sp. kku-s2 for lipid production using molasses as a carbon substrate. Internat. Conf. on Food Engin. and Biotech. IPCBEE vol. 9

Scarsella, M., Belotti, G., De Filippis, P., Bravi, M., 2010. Study on the optimal growing conditions of Chlorella vulgaris in bubble column photobioreactors. Paper prepared by the Dept. of Chem. Engin. Mater. Environ., Sapienza Uni. of Roma.

Sahoo, D., Elangbam, G., Devi, S.S., 2012. Using algae for carbon dioxide capture and bio-fuel production to combat climate change. Phykos 42 (1), 32-38.

Torres, H., 2013. On the growth of Chlorella vulgaris for lipid production. Poster presentation at the University of Puerto Rico.

Recommendations and Future Plans

New ideas to consider:– Fed batch systems (dynamic rather than static) will enhance

growth and increase yield (e.g. remove old medium and infuse fresh growth medium).

– Increase the carry capacity will increase maximum yield.– Applying a buffer to control pH fluctuations will allow higher

flow rates of CO2 gas and thus enhance growth.– Measure and control growth rates to guard against

overshoots and establish a maximum yield capacity (MYC).

Final Words and Acknowledgments

Small steps and small improvements, taken in the right direction, will sooner or later take you where you need to be.

Special thanks to the all the members of the departments of chemistry and biology who had a hand to play in this endeavor.