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CULTIVATION AND OPTIMIZATION OF BIOMASS PRODUCTION OF SPIRULINA

PLATENSIS USING ORGANIC SUBSTRATES

Article  in  Journal of Natural Remedies · January 2020

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 JNROnline Journal Journal of Natural Remedies
ISSN: 2320-3358 (e) Vol.21,No.5,(2020)
ISSN: 0972-5547(p)

CULTIVATION AND OPTIMIZATION OF BIOMASS PRODUCTION OF

SPIRULINA PLATENSIS USING ORGANIC SUBSTRATES
Sukanya.A* and David Ravindran.A
*Research scholar Department of Biology, The Gandhigram rural institute deemed to be
university, Gandhigram. Tamilnadu ,India.
Professor, The Gandhigram rural institute deemed to be university,
Gandhigram.Tamilnadu ,India.

Abstract: The present study to reduce the cost of cultivation expensive because the higher
concentration of sodium bicarbonate and other nutrition contents. Sodium bicarbonate was
added add 20-40 % such as( 3.36-6.72 g/l) helps to maintain the alkali pH, other nitrogen and
phosphate could be replaced by the low cost organic oil cake filtrate medium. The maximum
biomass production was observed 10th day of the cultivation, 5th experimental run,yield of
biomass and protein1.46g/100ml and 61.87w/w-1 ,all the seven factors statistically significant(p<0
0.05) performing the good growth in the all the experimental treatments.in Compared to the
standard Zarrouk media cultivation this low cost organic cultivation has been used as cost effective
and eco friendly medium for the cultivation Also this statistical optimization will also helps the
large scale biomass production for commercial level.More than 13 years of research gap between
the cultivation of Spirulina in organically,the present study fully focused on the organic materials
for the cultivation.
Keywords: Spirulina, Sodium bicarbonate, oil cake filtrate, optimization, biomass.

1. Introduction
Microalgae areoneoftheprospectivesources of chemical and bioactive compounds.Arthrospira
platensis(Spirulina platensis) is a filamentous cyanobacterium that is often called blue green algae. Due
to the high protein and bioactive compound S.platensis which is the most cultivated microalga
worldwide.Because of its composition, mainly which is rich in proteins (50-70%), composed of
essential amino acids, vitamins(includingB 12),polyunsaturated fatty acids, pigments,
and phenoliccompounds.Spirulina has been promoted as a food supplement[10].Spirulina platensis
cell extracts are shown potential bioactive properties such as moderate the blood cholesterol,
preventing cancer, stimulate the immune system to reduce the nephrotoxicity of
pharmaceuticals.Those types of bioactive properties have been recognized to
distinct compounds viz.,Phycobiliproteins, phenolic compounds, organic acids, beta carotenes,
polyunsaturated fatty acids, polysaccharides[12], and [6].Based on the literature many studies on
the cultivation of Spirulina in different agriculture and industrial waste effluents like kitchen
wastewater [7],swine wastewater [5], agro-industrial wastes and wastewater from agro industrials
[4].But there are only a few studies were done on the cultivation of Spirulina inorganically.[2]
proposed that the production of Spirulina sp in brain wastewater from the pickle industry.
[1]reported that cultivation(SCP) of Spirulina from rice mill waster water for the production of
Chlorophyll. The production of Spirulina has been estimated between 3000-10000 tons per year,[9]
cultivation ofSpirulina in the effluent from the anaerobic digestion. [8] rejected brine from inland
used for desalination the purpose of fish farming, cultivation of Spirulina, and irrigation of
exploring shrub and crops. The microalgae biochemical composition mainly depends on the
culture medium, pH, temperature, light intensity, and environmental factors stress. Microalgae is
the potential source for the CO2 sequestration and pollutant uptake from industrial waste water is
another important factor that can be a solution for many environmental cleanups. In terms of low-
cost fermentation medium plays a potential role in bio process technology. It also gives high-
quality product yield and effective recovery. To this demand, the fermented medium developed
through the cheep agro- industrial by-products which have to provide good nutritional conditions

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required for the growth and synthesis of the targeted products.The main objective of this study
was to optimize the organic media composition for the cultivation of Spirulina biomass production
and enhances the nutritive values of the biomass composition.
2. Materials and methods
Primary studies of biomass production Cultivation and Production of S.platensis viz different organic
oilcake filtrate medium was used fig (1)shows that the preliminary studies .During the primary cultivation oil cake
filtrate medium
supports the S.platensis growth and shape of the algae throughout the cultivation period

2.1. Preculture of Spirulina

S.platensis strain was obtained from the Department of Biology Gandhigram University, allow to
grown in Zarrouk media under the greenhouse condition and maintained as the mother inoculum
for the throughout cultivation period. The initial concentration of the mother inoculums was
measured at 560 nm. The culture medium was maintained at pH 9.0-9.5, and the light intensity of
the culture was maintained a 2700 Klux.

2.2.Measurement Of Growth Rate:

The value X1 and X2 is the optical density measured at absorbance 560 nm in spectrophotometer
with the time interval of 10 days represented by t1 and t2. The specific growth rate of algae
calculated by the following formula
𝑋2−𝑋1
𝜇 = ∫ 𝑡2−𝑡1 -------------(1)

2.3.Micro and macro nutrient analysis

The oil cake filtrates were analyzed essential micro nutrients such as calcium, potassium,
phosphorous through flame photometer (Systronics 129) and nitrogen, nitrite, phosphorous
contents were also analyzed.
Table:I. Analysis of micro-nutrients Sodium, Potassium, Calcium, the content available
in fermented de-oiled cake filtrate.

Table: II. Estimation of nitrogen content in de-oiled cakesby using the Kjeldahl method

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Table.I.and II showed that the micro and macro nutrient essential fro the growth of Spirulina.Thi
s present Study the substrates having the required amounts of nutrients. The availability of
nutrients contents were represented.
3. Result and Discussion
This statistical optimization method were used to optimize the organic media,there are seven
independent factors that enhance the biomass production of S.platensis such as full factorial design
was use with 8 experimental runs all the runs were duplicated . This full factorial design,this
experimental model was used to find out the potential factor significantly enhance the biomass in
S.platensis. Table (6) showed that the coded values of the the factor . The coded values are fixed
thought the preliminary studies Fig(3.1) showed and the coded values of the levels of the
independent factors are represented in table(III).During the organic cultivation all the algal culture
were maintained their spiral shape throughout the cultivation period.Fig.(2) showed that the spiral
shape of the S.platensis in 40X .Fig(3) SEM image of the S.platensis.

Table:III.Experimental independent variables at two levels used for the cultivation of

S.platensis using fractional factorial design

“-1 corresponds to the lowest value and “+1 corresponds to the highest value of the input
parameter range. Factor 1-5 represents in ml,6-7 represents “-1” corresponds to the minimum
value and “+1” corresponds to the maximum value of the input parameter rang

Fig.1.Preliminary studies of S.platensis in organic media Fig.2. Microscopic observation of

S.platensis under 40X

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Fig.3.SEM image of S.platensis in optimized media Fig.4. Wet biomass from organic media
Table.4 showed that the factorial design of the mediain different combination at different levels.In
the full factorial values are always lies between from 0%-100% . The results of the present study
determination of coefficient values of the biomass production was 0.996-and the adjusted R2 was
0.992.That above recorded results are closely related to the 100% it represents the significance of
the experimental model, the present study experimental model is highly significant. Figure .5shows
that the factors influences the biomass production of the S.platensis. The factors like
groundnut,coconut and neem cake filtrate concentration showed red and orange plot in the graph
indicates the significant yellow plot in the graph it indicates they are and non significant.
Table:IV.Fractional factorial design for the evaluation of independent variables with
coded values along with the growth response

From the experimental results runs of 5,6,13gives higher biomass production and proteinconcent
rationsuchas1.46,1.08,1.44gm/100 ml and 61.87, 60.43, 60.07 % of protein were observed. To
reduce the cost of the medium the present study attempts the oil cakes were chosen.The highest
optimum conditions were obtained from the B and C combination such as neem and coconut oil
cake medium has been fit for the highest biomass production and protein content. The highest
production was occur due to the combination of highest nitrogen (neem) and lowest nitrogen
(coconut) content in the media composition.Which enhances the biomass production and

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protein. Neem and Groundnut are rich in nitrogen[2] designed that the two organic media with
the combination of Neem cake and groundnut cake the highest biomass production was than
recorded in neem cake media 45±3%
Table : V.Regression statistics and Analysis of variance (ANOVA)for the Experimental
results fractional factorial design used for the production of Spirulina biomass
and protein content.

S-Significant values, SS- Sum of squares, MS - Mean square, F -Fisher value, P-value- the level of
significance, df- degree of freedom. a p-value less than 0.05 are considered to be significant, *- non
significant.(Table.3) showed that the Regression statistics and ANOVA for the experimental data,
and level of confirmation fit for the cultivation also represents the effect of the
experiment.(Table.4) showed that the coded coefficient of constant variables of the independent
variables. All the factors showed that the significant results.(Table.5) represent that the ANOVA
for the code values
Table.VI. Coded Coefficients of constant variables
Coefficient Standard error T P
Constant 0.86 0.008 112.05 0.063
9
A 0.05 0.008 6.592 0.078
B 0.19 0.008 24.821 0.142
C 0.15 0.008 19.287 0.011
D 0.02 0.008 3.011 0.017
E 0.04 0.008 5.615 0.001
F 0.07 0.008 8.708 0.019
G 0.20 0.008 25.960 0.023
Table.VII. Analysis of variance of independent variables
ANOVA Df SS MS F P
Regression 7 1.72 0.25 260.26 0.39
Error 8 0.01 0.00
Total 15 1.73
S=0.031.R2=0.992 R2 Adj=0.992
The analysis of variance of the model showed that low P values (< 0.0001) and high F-values for
these responses confirm that the developed models were remarkably significant. Moreover, the

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Vol. 21, No. 5,(2020)

insignificant lack of fit (p > 0.05) and the high values of R2, adj-R2, and pre-R2 apparently
demonstrated that the second-order regression could establish a good correlation between the
responses and the independent variables.Table3.6expressed that the combination of the factor
which supports the Spirulina cultivation, among the seven factor the only the three factors were
effectively supports the cultivation.
Table.VIII. Two way Analysis of variance of (ANOVA) interacting factors
Std. err T P Significant
Constant 0.86 0.008 112.059 0.000 ***
AxB -0.15 0.008 -19.287 0.000 ***
AxC 0.19 0.008 24.821 0.000 ***
AxD 0.04 0.008 5.615 0.001 ***
AxE -0.02 0.008 -3.011 0.017 ***
AxF -0.20 0.008 -25.960 0.000 ***
AxG 0.07 0.008 8.708 0.000 ***
BxC -0.05 0.008 -6.592 0.000 ***

Anova Df SS MS F P
Regression 7 1.72 0.25 260.26 0.43
Error 8 0.01 0.00
Total 15 1.73
S -0.031 , R²-0.996,R²adj-0.992

Fig. 5. Shows that the factors influences the biomass production of the S.platensis
4. Conclusion
In the last 50 years to huge attempts have been made to exploit microalgae on a technological scale
as a source of food, feed lipid, vitamins, fertilizer, pharmaceutical, and other specialty chemicals.
Due to the vast application, the Spirulina culture was grown in normal cultivation. The statistical
method is one of the efficient tools for optimization study.
Statistical method is a very good tool for the optimization of biotechnological processes involving
microorganisms, for this study, the influence of six nutritional factors on biomass production of
Arthrospira sp. had been tested with only 16 runs with the consequent saving time and

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cost.Compared to the zarrouk media is RS.10. appropriate production cost was of one liter media
but the organic media cost is RS.3 to 3.5
5. Acknowledgement
The authors are thankful to Department of Biology, Gandhigram Rural Institute-Deemed
University, Gandhigram, Tamilnadu, India for providing laboratory facility to the entire research
work.
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