Presented by

Niyamat M PanjeshaPune University, Pune.

AProject Report

OutlineAim and ObjectivesIntroductionMaterial and Method Result and discussionConclusionReferences

Aim & ObjectivesAim

The aim of study was “Cultivation of Spirulina spp. on Sugar Effulent for Production of Phycocyanin and an Efficient Method for Extraction of Phycocyanin.”

ObjectivesTo 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.

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.

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

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.

3. Phycocyanin

The Spirulina is an excellent source of phycocyanin , The protein fraction may contain up to 20% of phycocyanin

Phycobiliproteins

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.

Materials & Methods

Characterization of sugar effluent.

Micro organism and culture condition.

Growth Analysis of Spirulina on sugar effulent.

Phycocyanin extraction from Spirulina.

1. Characterization of sugar effulentThe 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.

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).

3.Production of Phycocyanin on sugar effulentThe 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.

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.

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

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.

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.

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.

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.

Phycocyanin extraction. Drying of Phycocyanin.

ConclusionFresh 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.

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.

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