Background:

Spirulina is a blue green algae of particular value to rural communities in developing countries for its high nutrient content. This algae is currently grown in small scale farms and distributed to local women and children to treat malnutrition. However much of the algae must be sold to higher markets, the profits of which are used to cross keep the process economically sustainable. The two major costs of the process include manual labor which is mostly used for tank agitation and from nutrient media ingredients. The subject of this study is the nutrient media used in these small scale farms and potential formulations that may reduce cost while maintaining nutrient content.

Purpose:

The purpose of this study is to formulate different growth medias for spirulina platensis cultivation and evaluate those medias against a control for biomass yield, and nutrient content.

Materials:

Chemicals

Table 1: Chemical stocks and resources requested

Active Component / Total g/l/component / Request Weight (g)
Sodium bicarbonate(g/L) / 49.1 / 150
Sodium Carbonate (Soda Ash)(g/L) / 5 / 15
Ethylenediaminetetraacetic acid / 0.08 / 1
Magnesium sulfate, crystalized, (7 H2O)(g/L) / 0.9 / 5
Monoammonium Phosphate, chrystalized(g/L) / 0.1 / 1
Urea(g/L) / 0.108 / 1
Potassium Nitrate / 2 / 5
sodium nitrate / 6 / 20
Ammonium Nitrate / 0.353 / 1
Sodium Chloride, crude(g/L) / 8 / 20
Potassium sulfate, crystallized(g/L) / 3 / 10
Muriate of potash (Potassium Chloride) / 0.898 / 5
Di-potassium Hydrogen Phosphate / 2 / 10
Ferrous Sulfate(g/L) / 0.055 / 1
Lime(g/L) / 0.02 / 1
Calcium Chloride / 0.05 / 1
Single Super Phosphate / 1.25 / 5
Boric Acid / 1.43 / 5
Magnessium(II)Chloride Tetrahydrate / 0.905 / 5
Zinc Sulfate Heptahydrate / 0.111 / 1
Sodium Molybdate / 0.00885 / 1
Copper(II)Sulfate / 0.0395 / 1

Equipment

Preculture and culture

Small Scale Photo bioreactors

Thermometer

4, 2L Bioreactor Jars

1, 500mL Bioreactor Jar

Air Pump and tubing

Spectrophotometer

Spirulina culture

pH Meter

Lux Meter

Harvesting

Filter Paper

Cheese cloth

Funnel

VWR dryer

Testing

Scale

Methods

Media Formulation

Cow Dung Ash Media

  1. Dry cow dung
  2. Burn cow dung
  3. Filter cow dung ash through a fine mesh filter to remove the unburned solids
  4. Mix the ash with the designated volume of water and let sit for 2 hours
  5. Filter through fine mesh and then through filter paper

Rice Mill Effluent

  1. Soak brown rice for 24 hours
  2. Strain soaking water and save
  3. Steam rice for 20 minutes
  4. Combine residual steam water with soak water, use as rice mill effluent

RMEM, RCM, ZarroukMedia, AntennaMedia, and A5 Micronutrient Solution

  1. Weigh out each component, refer to Table 2 for component weights
  2. Add components to a 1L beaker, acquiesce to 500mL with desired solvent
  3. Place graduated beaker on a stir plate, mix with stir bar
  4. Transfer mixed solution to a 3L container, acquiesce to 2L

Table 2: Component composition of Spirulina Medias

Atoms Provided / Active Component / 2L Antenna Media / 4 L x Zarrouk / 2L RME / 2L 20%CDAM 80%CFTRI / 2L RCM / 1L A5 Solution
Carbon / Sodium bicarbonate(g/L) / 2 / 67.2 / 20 / 9 / 33.6
Sodium Carbonate (Soda Ash)(g/L) / 10
Ethylenediaminetetraacetic acid / 0.32
Magnesium / Magnesium sulfate, crystalized, (7 H2O)(g/L) / 0.4 / 0.8 / 1
Nitrogen / Monoammonium Phosphate, chrystalized(g/L) / 0.2
Urea(g/L) / 0.04 / 0.176
Potassium Nitrate / 4
sodium nitrate / 10 / 4 / 3
Ammonium Nitrate / 0.706
Conductivity / Sodium Chloride, crude(g/L) / 10 / 4 / 2 / 2
Potassium / Potassium sulfate, crystallized(g/L) / 2 / 4 / 2
Muriate of potash (Potassium Chloride) / 1.796
Di-potassium Hydrogen Phosphate / 2 / 0.6 / 2.4
Iron / Ferrous Sulfate(g/L) / 0.01 / 0.04 / 0.08
Calcium / Lime(g/L) / 0.04 / 0
Calcium Chloride / 0.16 / 0.02
Single Super Phosphate / 2.5
Micro Nutrients / Boric Acid / 1.43
Magnessium(II)Chloride Tetrahydrate / 0.905
Zinc Sulfate Heptahydrate / 0.111
Sodium Molybdate / 0.00885
Copper(II)Sulfate / 0.0395
Cow Dung Ash / 80
Solvents / Sea Water
A5 Solution
Rice Mill Effluent / 2 L
CDAM / 400 mL
Water / 2 L / 1600mL / 2 L / 1 L

Preculture

  1. Fill a 500mL bioreactor jar with 450mL of Zarrouk media
  2. Inoculate flask with 45mL of living Spirulina
  3. Grow under 12:12 light, dark time ratio, with day time bubble agitation.
  4. Measure OD at 560nm at the same time daily, with a target OD of 0.8-1

Culture

  1. Fill a 2L bioreactor jar with 1500mL of each media to be tested
  2. Inoculate with 10% inoculum, approximately 150 mL
  3. Grow under 12:12 light, dark time ratio, with day time bubble agitation.
  4. Measure OD at 560nm at the same time daily
  5. Harvest algae when it reaches lag phase

Harvesting

  1. Remove jars from each bioreactor
  2. Filter contents through a funnel lined with cheese cloth
  3. Blot the algae dry with filter paper
  4. Place algae in VWR dryer to be dried
  5. Store algae in air tight container

Testing

  1. Weigh the harvested and dried algae
  2. Perform further testing…

Results

Preculture Parameters / Inoculation Parameters / Flask 1 / Flask 2 / Flask 3 / Flask 4 / Flask 5 / Flask 6
Media Type / Media Type
Media Volume / Media Volume
Temp / Temp
Inoculation time / Inoculation time
Inoculation date / Inoculation date
Lux / Lux
pH / Initial pH
Initial OD / Initial OD
Final OD / Final OD
Harvest time / Harvest time
Harvest date / Harvest date
Optical Density Table
Date / Time / Zarrouk / Antenna / RME / RCM / 20%CDAM / FM
T=0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
Day 14
pH Table
Date / Time / Zarrouk / Antenna / RME / RCM / 20%CDAM / FM
T=0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
Day 14
Temp Table
Date / Time / Zarrouk / Antenna / RME / RCM / 20%CDAM / FM
T=0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 10
Day 11
Day 12
Day 13
Day 14
Dry Weight
Zarrouk / Antenna / RME / RCM / 20%CDAM / FM
Volume Harvested
Dry Weight

Analysis Plan

  1. Plot each cultures OD readings vs. time against one another
  2. Compare dry weight values in a bar chart
  3. Compare observations concerning growth patterns
  4. Verify that pH and temperature values stayed consistent between cultures
  5. Save samples of the spent and fresh media for nutrient analysis
  6. Submit algae for Vitiman A, Zink, Iron, protein, and lipid content characterization