On the Growth of Chlorella vulgaris for Biofuel Production

Students: Barnes, Joseph, A.Garcia, Cynthia, N.Perez, Lirey, J.

Advisor: Torres, Hirohito, PhD, PE

Physics and Chemistry Department

Industrial Chemical Processes Technology

(Accredited by ABET)

Twelfth Undergraduate Research Forum

May 22, 2015

UPR - Arecibo

Introduction

Renewable Energy from Biofuel and Why We Should Use Microalgae to Get it

Biofuel Leads to Global Improvements

The demand for energy is increasing world wide as the population grows and countries undergo technological development.

A finite supply of fossil fuel cannot sustain an infinite demand for energy.

Producing fuel from biomass is a form of renewable energy, a means of meeting ever-increasing demands.

Biofuel Leads to National Improvements

Independence from the oil market and protection from its ripple effects.

A secure energy source brings stronger national security.

Switching from importing fuel to exporting fuel leads to a boon in the domestic economy.

Using Biomass from Microalgae as Source for Biofuel

Reaches an optimal harvesting stage within days instead of months, providing a high yield of biomass per acre of land.

Promises a high output of biofuel with minimal use of arable land, reducing competition between food and fuel, and thus reducing costs.

Suitable for establishing and sustaining a carbon neutral process.

Can engage a heterotrophic metabolism, consuming organic substances and serving as a means of bioremediation, coupling one environmental benefit with another.

Chlorella vulgaris

Freshwater unicellular algae with chlorophyll pigments -a and -b, which enable oxygenic photosynthesis.

Requires only water, CO2, and some minerals for growth.

Possesses a lipid content as low as %15 or as high as 40% or higher, depending on stress conditions, such as available nitrogen.

Contains other unique sugars and proteins that are useful to other industries, making C. vulgaris very desirable for its multifaceted usefulness.

Using Biomass from Microalgae as Source for Biofuel

Chlorella vulgaris – Multifaceted Usefulness

Industrial uses: Bioremediation (via heterotrophic metabolism).

Production of ethanol from corn and switchgrass plant (via cellulose degrading enzymes).

Medicinal uses: Removing heavy metals from the body (possesses

chelating agents). Helps restore elasticity to the skin, diminishing

wrinkles and stretch marks (contains active ingredient, dermachlorella D/DP).

Reduces detrimental effects caused by exposure to harmful radiation (e.g. UV radiation).

Chlorella vulgaris – Multifaceted Usefulness

Objectives

Long-term and Short-term

Long-term Objectives

Attain a biomass density adequate for lipid extraction and biodiesel production: biomass density of 1.0 g/L or higher and specific growth rate of 0.50 day-1.

Encourage the establishment and expansion of microalgal farms to produce biofuel.

Buy less foreign fuel, and invest more in the economy.

Short-term Objectives

Enhance biomass production by implementing a fed-batch system.

Enhance biomass production by use of higher light intensities and mono-colored light sources (650 nm and 475 nm).

Explore other means of enhancing biomass densities and specific growth rates.

Materials and Equipment set-up

Materials and Inorganic Substrates

Graduated bottles. Erlenmeyer Flasks. LED growth lamps. Aquarium fluorescent

lamp. Commercial-grade

aquarium pumps. Glass bright-line

hemocytometer. Optical microscope. Light meter. LED lights with

changeable wavelengths. Spectrophotometer Light Meter

Tropical-brand 20-20-20 (Very economical and cost-efficient fertilizer).

OrchidPlus-brand 20-14-13 fertilizer (Very economical and cost-efficient fertilizer).

MgSO4

NaCl NaNO2

Materials and Inorganic Substrates

Materials and Inorganic Substrates

Procedures Implemented and Results Recorded

Challenge

Exploring any potential means of enhancing the carry capacity of indoor photobioreactors in order to improve biomass density.

Method 1

Three groups, two experimental and one control, each in duplicate.

Initial substrate concentrations for all photobioreactors:

Nitrogen (ammoniacal and organic) – 0.15 g/L Phosphate – 0.15 g/L Potassium (Potash) – 0.15 g/L

Aeration - ~613 ml/min. Light Source – White light; the average light intensity incident

on the surface of the photobioreactors was 3283 lux. (Summer sunlight >100,000 lux)

Photoperiod - 12hr/12hr light/dark cycle. Duration – 24 days Exp. Group I – Light feeding, receiving a total of 0.45 g N, K,

P2O5. Exp. Group II – Heavy feeding, receiving a total of 0.90 g N, K,

P2O5.

Results M-1: Maximum Cell Concentrations & Biomass Density

0.8175

1.344

1.079

0.295

0.24

0.085

0 0.5 1 1.5

Control Group

Exp. Group I

Exp. Group II

Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

Method 2

Three groups, two experimental and one control, each in duplicate. Initial substrate concentrations for all photobioreactors:

Nitrogen (ammoniacal and organic) – 0.20 g/L Potassium (potash) – 0.14 g/L Phosphate – 0.13 g/L MgSO4 – 0.335 g/L NaCl – 0.125 g/L NaNO2 – 0.250 g/L Micronutrients – Trace amounts

Aeration - ~613 ml/min. Light source – 650 nm and 475 nm light; the average light intensity

incident on the surface of the photobioreactors was 3071 lux. Photoperiod - 12hr/12hr light/dark cycle. CO2 supplementation - 5 to 10% of total volume of gas injected. Duration – 21 days. Exp I – Light feeding, receiving a total of 0.07 g N, 0.11 g MgSO4. Exp II – Heavy feeding, receiving a total of 0.14 g N, 0.23 g MgSO4.

Results M-1 & M-2: Maximum Cell Concentrations & Biomass Density

1.436

2.119

2.373

0.8175

1.344

1.079

0.57

0.52

0.51

0.295

0.24

0.085

0 0.5 1 1.5 2 2.5

M-2: Control

M-2: Exp I

M-2: Exp II

M-I: Control

M-I: Exp. I

M-I: Exp. II

Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

Method 3 – Increasing Light Intensity

Three groups, two experimental and one control, each in duplicate.

Initial substrate concentrations, Aeration, Photoperiod, CO2 supplementation, Duration, Feeding portions – Same as Method 2.

Light source - Same as Method 2, but with higher intensity at 6217 lux.

Results M-1, M-2 & M-3: Maximum Cell Concentrations & Biomass Density

1.436

2.119

2.373

0.8175

1.344

1.079

0.4

0.35

0.46

0.57

0.52

0.51

0.295

0.24

0.085

0 0.5 1 1.5 2 2.5

M-3: Control

M-3: Exp. I

M-3: Exp. II

M-2: Control

M-2: Exp. I

M-2: Exp. II

M-1: Control

M-1: Exp. I

M-1: Exp. II

Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

Results M-3: Alternative explanation for drop in biomass density

Interval between last dose and biomass reading:

M-2: 12 days.M-3: 4 days.

Longer starvation period, more cell growth, more biomass.

Fed-Batch Redemption

M-3: Re-evaluated

Left the experiment running.Analyzed the relationship between

biomass density and time up till last biomass reading.

Results M-3: Biomass density (g/L) versus Time (days)

0

0.1

0.2

0.3

0.4

0.5

0.6

1 3 5 7 9 11 13 15 17

Time (Days)

Bio

mas

s D

ensi

ty (

g/L

)

Control

Exp. Gr. I

Exp. Gr. II

Results M-3: Calculating Biomass Density With More Precision

Starved all groups for 8 days. Method relied on the natural

tendency of C. vulgaris to precipitate, fall out of solution, when in stagnant phase.

Weighed entire biomass and divided by 0.500 L.

Results M-3: Biomass density (g/L) versus Time (days) – Adjusted

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

1 3 5 7 9 11 13 15 17 19 21 23 25

Time (days)

Bio

mas

s D

ensi

ty (

g/L

)

Control

Exp. Gr. I

Exp. Gr. II

Results M-1, M-2 & M-3: Biomass Density - Adjusted

0.34

0.48

0.58

0.57

0.52

0.51

0.295

0.24

0.085

0 0.2 0.4 0.6 0.8 1

M-3: Control

M-3: Exp. I

M-3: Exp. II

M-2: Control

M-2: Exp. I

M-2: Exp. II

M-1: Control

M-1: Exp. I

M-1: Exp. II

Biomass Density (g/L)

Conclusions

Providing additional nitrogen (ammoniacal and organic), potassium, and phosphate alone did not lead to any substantial increase in biomass density.

By modifying the method for reading biomass density, we can see that a regulated fed-batch system can augment carry capacity.

Recommendations & Future Plans

Analyze other specific variables for possible enhancements: Light intensity (Redo) Photoperiod (e.g. 16hr/8hr light-dark cycle) Surface area to volume ratio.

Replicate conditions for reaching 0.60 g/L, and find a consistent result.

Inoculation: Day 1

Feeding – 18 d Starvation 3 d

Precipitation 5 d

Extraction: Day 26

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.

All about algae (2015). Algae Biomass Organization. Retrieved May 26, 2015, fromhttp://allaboutalgae.com/why-algae/

Christi, Yusuf, 2007. Biodiesel from microalgae. Research review paper prepared at the Institute of Tech. and Engineering, Massey University, New Zealand. Biotechnological Advances 25 (2007) 294-306.

Additional Observations: Effects of mono-color light on Cell Conc.

Initial substrate concentrations, Aeration, Photoperiod – Same as Method 1.

Duration – 7 days Light source - Intensity of light incident on the

surface depended on wavelength: Red – 638 lux Blue – 653 lux Green – 977 lux

Additional Observations: Effects of mono-color light on Cell Conc.

7.45

4.36

11.17

0

2

4

6

8

10

12

Cel

l C

on

cen

trat

ion

(10

^6

cell

s/m

l)

475 nm

510 nm

650 nm

Absorbance (A) versus Wavelength (nm)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

370410

450490

530570

610650

690730

Wavelength (nm)

Ab

so

rba

nc

e (

A)

Additional Observations: Surface Area to Volume Ratio.

Initial substrate concentrations, Aeration, Light Source, Photoperiod – Same as Method 2 & 3.

Duration – 14 days.

Reactor A – S.A: 332.9 cm2; Vol: 242.1 ml Ratio: 1.375 cm2/ml

Reactor B – S.A: 572.3 cm2; Vol: 880.0 ml Ratio: 0.650 cm2/ml

Additional Observations: Surface Area to Volume Ratio.

1.13

0.6328

0.22

0.09

0 0.2 0.4 0.6 0.8 1 1.2

1.375 cm^2/ml

0.650 cm^2/ml

Cell Concentration (10^7 cells/ml) Biomass Density (g/L)

Questions?