Qualitative Media Analysis
Introduction
Algae species such as Spirulina platensis are high in proteins, essential oils, and micronutrients.
Cultivation of Spirulina is a means to treating malnutrition especially in rural communities with a lack of
access to alternative treatment. The nutrients required for algae growth include carbon, nitrogen, and
potassium which can be sourced from a multitude of different chemicals and fertilizers. Additionally
magnesium, iron, calcium, and phosphorus are beneficial components when attempting to treat
malnutrition due to common deficiencies in these compounds.
The liquid growth media used has a huge impact on the growth behavior and nutrient composition of
the algae. This evaluation examined nine different growth medias from five different papers, their
composition, and resulting performance relative to the control used in each study. The goal is to
identify the best performing medias and cross compare them based on component identity and
concentration. Special attention will be paid to the top four biomass yielding medias, Rice Mill Effluent
Media (RME), 20% Cow Dung Ash Media (CDAM), Enriched Seawater Media (ESWM) and Reduced Cost
Media (RCM). In most of the studies evaluated the control used was Zarrouk media which has been the
standard media for spirulina cultivation for the last 50 years.
Carbon Source
All medias evaluated use Sodium Bicarbonate or baking soda as a source of carbon, the Lewis structure
of which can be seen in figure 1. Only the Antenna Media uses a second source of carbon, Sodium
carbonate or soda ash seen in Figure 2, other than atmospheric CO2 which is assumed to be present in
all medias. It was found that atmospheric CO2 is not a preferred source of carbon for Spirulina but
rather that at higher concentrations it hinders growth.
Figure 1: Sodium Bicarbonate
Figure 2: Sodium Carbonate
The concentrations of the aforementioned carbon sources vary in a range of 1 g/L-16.8 g/L between
medias. However it can be seen that medias with a sodium bicarbonate concentration of greater than
10 g/L have the highest dry biomass yield relative to their respective controls with the exception of the
20% Cow Dung Ash Manure (CDAM) with a Sodium bicarbonate concentration of 4.5 g/L which may be
supplemented by organic carbon provided by the cow dung ash.
Magnesium Source
The most common source of magnesium among the medias analyzed is Magnesium
Sulfate seen in Figure 3 which is present in six of the nine analyzed varying from
0.15g/L to 1.2g/L. Among the top three biomass yielding medias Magnesium is
present at a high concentration of 1.2g/L in 20% CDAM compared to the control
Zarrouk media at a concentration of 0.2g/L. The other two medias have complex
sources of magnesium from the rice mill effluent water containing 43.78mg/L and
muriate of potash with an unknown concentration of magnesium in the RCM.
Figure 3: magnesium Sulfate
Nitrogen
There were five discrete sources of Nitrogen used among the nine medias including Monoammonium
Phosphate, Urea, potassium nitrate, sodium nitrate, and ammonium nitrate seen in figures 4-7
respectively.
Figure 4: Monammonium
Phosphate
Figure 5: Urea
Figure 6: Potassium
Nitrate
Figure 7: Sodium Nitrate
Figure 8: Ammonium
Nitrate
Sodium nitrate is present in six out of nine medias ranging from 1.5g/L to 2.5g/L. RCM uses two sources
of nitrogen, Urea and ammonium nitrate, at 0.088g/L and 0.353g/L respectively. The antenna media is
the only one to use monoammonium phosphate and potassium nitrate, at 0.1g/l and 2g/L respectively.
The antenna media also uses a 0.2g/L concentration of urea.
Conductivity/Sodium Ions
All medias contain some sodium chloride ranging from 0.5g/l to 5g/l. The top three performing medias
and the Zarrouk control media have concentrations of 1g/L of NaCl with the source of sodium in the
RME media coming from the rice mill effluent at 1g/L.
Potassium
There were four discrete sources of potassium used among the nine medias including potassium nitrate,
potassium sulfate, Muriate of potash, and Di-potassium hydrogen phosphate seen in figures 9-12
respectively.
Figure 9: Potassium Nitrate
Figure 10: Potassium Sulfate
Figure 11: Muriate of potash Figure 12: Di-potassium hydrogen phosphate
Potassium Sulfate and Di-potassium hydrogen phosphate are present in five out of nine of the medias at
concentrations varying form 0.5g/l to 1g/l and 0.1g/l to 0.5g/l respectively. The Antenna media uses
2g/l of potassium nitrate in addition to potassium sulfate. 0.898g/l of muriate of potash is the singular
source of potassium in RCM.
Iron
In those medias that contain discrete sources of iron, Zarrouk, Antenna,
20%CDAM, modified NRC and Offer media the source is Ferrous sulfate
seen in figure 13. The 20%CDAM media contains additional iron from the
cow dung ash which contains 4.88mg/L of iron. RME, RCM, and ESWM
don’t appear to have any significant source of iron. The resultant culture
in these medias would most likely not be sufficient in iron to benefit those
with deficiencies.
Figure 13: Ferrous Sulfate
Calcium
The three sources of Calcium include lime, calcium chloride, and single super phosphate seen in figures
14-16 respectively.
Figure 14: Lime
Figure 15: Calcium chloride
Figure 16: Single Super Phosphate
It appears this component is needed in only low amounts as the concentrations of the different calcium
sources range from 0.02g/L to 0.04g/L except for the complex source single super phosphate which is
present at 1.25g/L in RCM. The RME media also contains 50.36mg/L of calcium from the rice effluent.
Phosphorus
The components containing phosphorus include monammonium phosphate, phosphoric acid, and single
super phosphate.
Figure 17: Monammonium
Phosphate
Figure 18: Phosphoric Acid
Figure 19: Single Super Phosphate
Discrete phosphorus is only present in four of the nine medias. Of those only two contain single supper
phosphate with RCM at a concentration of 12.5g/L and Revised 6 at 1.25g/L. The Antenna media
contains 0.1g/L monammonium phosphate which may primarily serve as a nitrogen source rather than a
phosphorus source.
Complex
The complex medias include Sea water, Rice Mill Effluent and Cow Dung Ash Media the respective
composition of each can be seen in table 1.
Table 1: Complex media composition
Sodium
Sulfate
Nitrate
Calcium
Magnesium
Potassium
Carbon
PsO5
K2O
Zinc
Copper
Iron
Sea Water
11.8 (mg/L)
2.95 (mg/L)
400 (mg/L)
1.403 (mg/L)
Rice Mill Effluent
100.09 (mg/L)
2.5 (mg/L)
50.36 (mg/L)
43.78 (mg/L)
8.0 (mg/L)
CDAM
3.10 (mg/L)
0.17%
60 (kg/Ha)
1613 (kg/Ha)
3.76 (mg/L)
1.02 (mg/L)
4.88 (mg/L)
Discussion
Rice Mill Effluent media, 20% Cow Dung Ash Media, Reduced Cost Media, and Enriched Seawater media
all produced dry biomass within 10% of their relative controls. RME and 20% CDAM produced
approximately 10% more biomass than the control.
Rice Mill Effluent (RME) media only requires three additional components in addition to the RME
consisting of sodium bicarbonate as a carbon source, sodium nitrate as a nitrogen source, and dipotassium hydrogen phosphate as a potassium source. These are all components used in the Zarrouk
media but the nitrogen and potassium sources are different from those used in the Antenna media
The 20% CDAM has almost all the components of Zarrouk media of varying concentrations with the
exception of calcium derived from lime, and no ethylenediaminetetraacetic acid. This is because the
other 80% of the media is CFTRI a variation of Zarrouk media. The 20% CDAM contains a variety of
metals that are desirable for algae growth that are absent in the other medias.
The RCM was designed to decrease the cost of growing spirulina while maintaining the high productivity
achieved by Zarrouk media. Cost was reduced per 1000L of media from US$80 to US$13 while
maintaining 95.2% and 99% of Zarrouk media biomass and protein production respectively. This was
accomplished by replacing the relatively expensive nitrogen source sodium nitrate with a mixture of
ammonium nitrate, and urea and by providing potassium, calcium and phosphorus with the common
fertilizers muriate of potash and single super phosphate instead of potassium sulfate, and calcium
chloride. Antenna media differs in many components from both Zarrouk media and RCM.
ESWM media has the same three added components as RME media but with lower concentrations. In
their respective studies they were both compared to Zarrouk media as a control. However their
respective yields in terms of protein, chlorophyll, and lipid content relative to their control was
significantly different as seen in Table 2. This suggests that there are nutrients present in RME and
Zarrouk media that are absent in seawater that contribute to chlorophyll, lipid, and protein production.
Table 2: Comparison of RME to Enriched Seawater
Control (Zarrouk)
Dry Biomass (g/L)
2.92
Protein (mg/mL)
1.5
Chlorophyll (mg/mL)
0.27%
RME
3.245
1.9
0.35
% of Control
111.130137
0.362
0.334
92.26519337
126.6666667
64.6
60.1
93.03405573
132.0754717
26
23
88.46153846
Control (Zarrouk)
ESWM
% of Control
Lipid (mg/mL)
0.218
0.26
119.266055
6.5
6
92.30769231
Conclusion
The components of each of the nine medias were categorized based on the atoms they provide to the
metabolic pathway of Spirulina. It was observed that different compounds or cocktails of compounds
could be used to provide sufficient quantities of any one of the required atoms. However the way in
which these exchanges affect growth parameters and algae composition are not fully known.
The four medias considered strongly by this paper RME, 20%CDAM, RCM, and ESWM, all produced dry
biomass within 10% of that of the control for that respective study. RCM was formulated to cut costs,
while RME and ESWM minimize cost while also minimizing discrete added ingredients down to three.
20% CDAM was designed to replace a fraction of an existing media with almost free macro and micro
nutrients. The respective strengths of each media offer an opportunity to hybridize formulations to
minimize cost and complexity while increasing productivity and nutrient content.
The next step of analysis that is needed is to determine costs of the components of each of the four
aforementioned medias. The purpose of this cost analysis is to see if certain components that provide
carbon or nitrogen for example could be substituted by cheaper components of similar function from
other medias. It must then be confirmed that each component is locally available. Finally a new
combination of components will be formulated to decrease costs and number of ingredients while
maintaining that the ingredients are locally sourced, a Frankenstein media (FM) if you will.
The growth of FM will then be compared to that achieved by RME media, RCM, 20%CDAM, Zarrouk
media, and Antenna media. If possible the protein, lipid, chlorophyll a, carotenoid, zinc, and iron
contents will be quantified and compared.