1. Presented by
Niyamat M Panjesha
Pune University, Pune.
A
Project Report

2. Outline
Aim and Objectives
Introduction
Material and Method
Result and discussion
Conclusion
References

3. Aim & Objectives
Aim
The aim of study was “Cultivation of Spirulina spp. on
Sugar Effulent for Production of Phycocyanin and an
Efficient Method for Extraction of Phycocyanin.”
Objectives
To characterization of Sugar industry effluent.
To study growth of Spirulina spp. in the sugar effulent for
production of Phycocyanin.
To analysis efficiency of Phycocyanin extraction method.

4. Introduction
The sugar effulent rich in inorganic and organic nutrients
therefore it is favorable for the cultivation of Cyanobacteria
among them the Spirulina spp.
Moreover the nutrients found in the sugar effulent can be
can be converted into biomass and this biomass can be
harvested to the extraction of Phycocyanin. ( Kailas M.
Doke & Ejazuddin M. Khan., 2011).
Cultivation of Spirulina Spp. for production of
phycocyanin at large scale mostly carried out on Zarrouks
medium. Use of sugar effulent as a medium can be
beneficial.

5. 1. Characteristic of sugar effulent
Sr. No. Characteristic. Value
1 pH 3.5-4.0
2 Suspended Solids, mg/L 400-450
3 Total Dissolved Solids, mg/L 2000-2100
4 BOD, 5 days at 20 C, mg/L 1200-1500
5 COD, mg/l 2000-2500
6 Oil & Grease, mg/L 100-100

6. 2. Spirulina Spp.
Cynophyceae, filamentous,
Blue-green algae.
Spirulina spp. contain high
level of protein, pigments &
linoleic acid.
Easy reproduction and high
reproductivity (Kilic et al.,
2006).
Optimum temp. 30-350
C
and resistant to High pH.

7. 3. Phycocyanin
The Spirulina is an excellent source of phycocyanin , The
protein fraction may contain up to 20% of phycocyanin
Phycobiliproteins

8. Phycocyanin is composed of two a & b protein subunits of 17
KDa & 19 KDa respectively.
The absorption Spectrum of Phycocyanin is 615-618 nm.
Phycocyanin has been used mainly as a food pigment and
cosmetic, recently , it’s also has shown the anti-inflammatory
and anti-cancer properties.
In view of the multiple use of Phycocyanin we have
investigated different extraction procedures and effect of
processing methods on phycocyanin yield.

9. Materials & Methods
Characterization of sugar effluent.
Micro organism and culture condition.
Growth Analysis of Spirulina on sugar effulent.
Phycocyanin extraction from Spirulina.

10. 1. Characterization of sugar effulent
The effulent from a sugar industry located at
Bhavninagar, Indapur, Pune was collected.
Following parameters were analyzed by standard
protocols provided by VSI.
pH.
Electrical conductivity (EC).
COD.
BOD.
Nitrate.
Potassium.
Phosphate.

11. 2. Microorganism & culture medium
The microalgae Spirulina Spp. culture collected from,
Krushi Vigyaan kendra, babulgav, dist Ahemadnagar.
The preparation and maintenance of the inoculum
was accomplished using Zarrouk medium.
Sugar effulent itself, used as culture medium for the
cultivation of Spirulina spp. for the production of
Phycocyanin after the neutralized with lime (pH 7).

12. 3.Production of Phycocyanin on sugar
effulent
The spirulina culture was
grown on rotatary shaker at320
C in 250 ml conical flask.
The culture were grown for a
period of 5 days and growth
was monitored
spectrophotometrically at 660
nm.
Data of OD at 660 nm vs.
Culture time were plotted and
growth curve was made.

13. 4.Extraction of Phycocyanin from Spirulina
Seven different methods were used for extraction of Phycocyanin.
Drying methods.
Water bath drying.
Sun drying.
Oven drying
Wet cell methods.
Water extraction.
Freezing and thawing.
Homoginization.
Acid extract.

14. Formula
where PC is the phycocyanin concentration (mg/g)
OD615 is the optical density at 615-618 nm
OD652 is the optical density at 652 nm

15. Results
Sr. No. Characteristic. Value
1 pH 4.1
2 Electric Conductivity 1.57
3 BOD, 5 days at 20 C, mg/L 1200
4 COD, mg/L 2112
5 Nitrate 12.80
6 Phosphate 34.29
7 Potassium 91.00
1.Characteristics of sugar effulent:
The Physico-chemical properties of sugar effulent were analyzed.

16. Growth of
Spirulina
in hrs
Run 1 Run 2
OD. pH OD pH
0 0.201 7
0.218 7
24 0.592 7.8 0.586 7.6
48 0.846 8.5 0.835 8.4
72 1.244 9.2 1.192 8.9
96 1.342 10.7 1.294 10.4
120 1.287 9.7
1.138 10.1
2. Growth curve of spirulina.
The growth of Spirulina in Sugar effluent was analyzed.

17. 3.Phycocyanin extracted
by drying methods.
Three different drying methods were used for the
extraction of phycocyanin and following results
were obtained.
In waterbath drying method the yield of
phycocyanin was obtained 4.29 mg and 4.27 mg
per gm of dry biomass of spirulina.
In Sun drying method phycocyanin conc.. was 3.90
mg & 4.09 mg respectively in run1 & Run2.
In oven drying method the phycocyanin conc. Was
2.87 mg & 3.34 mg.

18. 4.Phycocyanin extracted
by wet methods.
Four different wet methods were used for the
extraction of phycocyanin and the following results
were obtained.
Water extraction methods resulted in 10.63 mg &
10.78 mg phycocyanin per 100 mg of wet cells in
Run1 & Run2 respectively.
In freezing and thawing methods the Conc. Of
phycocyanin obtained was 12.93 mg & 12.49 mg as
compared to Acid extraction method 8.65 mg &
7.49 mg of phycocyanin obtained in Run1 & Run2
respectively.
The homogenization method with highest
phycocyanin yield of 14.39 mg & 14.55 mg/ 100 mg
of spirulina.

19. Phycocyanin extraction. Drying of Phycocyanin.

20. Conclusion
Fresh biomass is best for phycocyanin extraction.
The significant loss of phycocyanin in dried samples could be due to
its peripheral position in phycobilisomes on the thylakoid membrane
and its sensitivity to temperature.
Homogenization method is rapid and showed highest efficiency
compared with other methods.
Cultivation of Spirulina spp. on sugar effulent for production of
phycocyanin will reduce the cost of culture medium .
It can prove an excellent source of income generation for the
industry.

21. References
 Benedetti S, Rinalducci S, Benvenuti F, Francogli S, Pagliarani S, Giorgi L, Micheloni M,
D’Amici GM, Zolla L, Canestrari F. 2006. Purification and characterization of phycocyanin
from the blue-green alga Aphanizomenon flos-aquae., Journal of Chromatography B., (833)
12–18.
 Bhaskar SU, Gopalaswamy G, Raghu R. 2005. A simple method for efficient extraction and
purification of c-phycocyanin from Spirulina platensis Geitler. Indian. J. Exp. Biol. 43, 277–
279.
 Boussiba, S., Richmond, A.: Isolation and purification of phycocyanins from the blue-green
alga SpiruIina platensis. Arch. Microbiol. 120, 155-159 (1979).
 C. C. Moraes, Luisa Sala, G. P. Cerveira and S. J. Kalil. : C-Phycocyanin extraction from
Spirulina platensis wet biomass. Brazilian journal of chemical engineering, vol 28, no. 01, pp.
45- 49, January- march, 2011.
 Eriksen NT. 2008. Production of phycocyanin – a pigment with applications in biology,
biotechnology, foods and medicine. Appl. Microbiol. Biotechnol. (80) 1–14.
 O’hEocha C. Spectral properties of the phycobilins. 1. Phycocyanobilin. Biochemistry 1963;2:375–82.

22. Thank You