FORMULATION OF COST EFFECTIVE RICE MILL
EFFLUENT MEDIUM FOR THE MASS PRODUCTION
OF SINGLE CELL PROTEIN (SCP)
AMALA.K AND N.RAMANATHAN
Department of Microbiology, Annamalai University,
Annamalai nagar, Tamilnadu-India.
Abstract:
Spirulina is a photosynthetic, filamentous, non differentiated, multicellular
blue green microalga that grows naturally in warm climate. The microalgae Spirulina is
a source of protein, which is used as a protein supplement for humans, chicks and also in
aquaculture. Spirulina platensis culture was isolated from two different locations
namely Puducherry and Thiruvannamalai and designated as S3 and S4. The growth of
S3 and S4 strains was estimated in Zarrouk's medium under laboratory condition.
Various parameters like optical density, cell population and biomass was estimated. The
effect of temperature and pH was determined for these strains S3 and S4 and highest
biomass was observed at 30°C of pH 9.5. A new rice mill effluent (RME) medium was
formulated for mass production of Spirulina by supplemented with various nutrients
(NaNO3, KNO3, K2HPO4 and KH2PO4) and other cost-effective chemicals. This newly
formulated RME medium generates valuable growth of Spirulina platensis. It is locally
available, eco-friendly and cost effective medium.
RME medium, nitrogen, phosphorus, cost effective, Spirulina, biomass.
INTRODUCTION
Spirulina platensis is a planktonic photosynthetic filamentous cyanobacterium that forms massive
populations in tropical water bodies which have high alkaline pH value up to 11.0. The cyanobacterium
Spirulina contains 74% dry weight of proteins, along with high concentrations of minerals, pigments,
unsaturated fatty-acids and vitamins (Cohen, 1997) because of which it is used as a dietary supplement,
nutrient source in food, feed and pharmaceutical industries especially in developing countries. It can grow
in a wide range of environments like soil, sand, marshes, brackish water, sea water and fresh water (Ciferri
et al., 1983).
Rice milling is a process of removing husk and part of the bran from paddy in order to produce
edible rice. Parboiled rice production generally requires a large amount of water for soaking of the paddy.
After soaking, the water is drained out. If this water is not properly treated could result in water pollution
due to high levels of organic material present in rice mill effluent waste water. This effluent has high BOD,
COD and organic contents mainly in the forms of carbon, thus having the potential to damage and
deteriorate the environment (Pradhan and Sahu, 2004). Therefore, it needs to be treated before disposal,
Literature reports indicate that biodegradation involving microorganisms is a suitable process for industrial
waste water treatments (Noorjahan et al., 2005).
Olguin et al., 2001 reported that Spirulina has potential to reduce BOD of high carbon containing
waste water due to its mixotropic nature. Rice mill effluent (RME) a rich source of starch and other nutrients
can support profuse growth and aid in the mass multiplication of Spirulina. In the present study, the potential of using RME safe for disposal into the environment by reducing its BOD and supplemented with
various concentration of N, P and Carbon nutrient sources and formulate low cost RME medium such a
process would also generate valuable Spirulina biomass and create eco-friendly environment.
METERIALS AND METHODS
Isolation of Spirulina platensis strains and characterized
The microalga Spirulina was isolated from Pondycherry and Thiruvannamalai and named as S3
and S4. These strains was characterized based on parameters like average no. of spirals, direction of helix,
distance between spirals, diameter of spirals, width, length and shape of the spirals, pH tolerance,
temperature tolerance and habitat. Morphological characters were observed under microscope by using
micrometric method. These strains were grown in Zarrouk's medium under laboratory condition at 30ºC
light chamber for 30 days. The effect of temperature of two strains was determined by 250 ml Erlenmeyer
conical flasks were used, containing 100 ml of the growth medium. Each flask was inoculated with 10 ml
(0.05 mg) of the pure culture of the organisms. Each temperature regime was in triplicates. The culture were
incubated in water bath at the appropriate temperature (25°C, 30°C, 35°C and 40°C) and the periodically
replenished with the growth medium to prevent drying up of the cultures. The effect of pH was determined
by 250 ml Erlenmeyer conical flasks were used each containing 100 ml of the growth medium. Each flask
was inoculated with 10 ml (0.05 mg) of the pure culture of the organisms. Each pH (9.0, 9.5 and 10.0) was
triplicate. The growth of Spirulina platensis was measured the parameters optical density, cell population
and dry weight. The exhausted broth was collected for analyses. One portion was used for determination of
dry cell mass concentration by optical density (OD) measurement at 560 nm using a calibration curve.
Optical density was measured by using a spectrophotometer. The dry weight was measured by 100 ml of
culture was sampled and filtered through what man No.1 filter paper and dried for 1 hr at 105ºC and
weighed prior to filtration. The filtered wet biomass was then washed with two volumes of distilled water,
dried as above and weighed. Cell population was estimated by direct microscopic count method.
No .of spirulina per ml of sample = average no.of spirulina cell x no.of microscopic
field per sq.cm x dilution factor.
= x x n x 100
The dry weight was measured by 100 ml of culture was sampled and filtered through what man
No.1 filter paper and dried for 1 hr at 105ºC and weighed prior to filtration. The filtered wet biomass was
then washed with two volumes of distilled water, dried as above and weighed. The biomass concentrations
in the cultures were determined through the cell weight measured by the method of Vonshak et al., 1982.
COLLECTION OFRICE MILLEFFLUENT
For laboratory experiment the rice mill effluent was collected from Parvathi rice mill from
Sethiyathopu, Cuddalore district, Tamilnadu. The physico-chemical characters of the effluent were
analysed and were presented in Table 2.
RME MEDIAFORMULATION
The collected effluent was filtered through what mann No.1 filter paper to remove the dust
particles. Four 250 ml conical flask were taken and 100 ml of rice mill effluent was added, in each conical
-1
flask various nitrogen (NaNO and KNO 1gL-1) and phosphorus (K HPO and KH PO 0.5 gL ) sources
3 3 2 4 2 4
were added separately, and add trace amount of CaCl , FeSO and EDTAin each flask, pH was adjusted with
2 4
10 g-1 NaHCO and Rice Mill Effluent (RME) broth was formulated. It was sterilized in an autoclave. The
3
S and S strains were inoculated to the medium and incubated for 30 days in light chamber, after 30 days
3 4
optical density value and dry weight was estimated. The highest growth was observed in both NaNO and
3
K HPO added media.
2 4
Mass production of Spirulina SCPin RME media compare with routine ZM media
The newly formulated Rice Mill Medium (RME) was prepared and transfer in 1 liter Erlenmeyer
conical flask, standard inoculums (50 ml) of two strains were separately inoculated and maintained at room
temperature in light chamber for 30 days. After 30 days of growth the parameters such as optical density,
cell population and dry weight were estimated by various methods are discribed earlier.
RESULTAND DISCUSSION
The isolated algal samples S3 and S4 srains general morphological characteristic like average
no.of spirals, direction of helix, distance between spirals, diameter, width, length and shape of spirals were
observed under microscope. And pH tolerance, temperature tolerance and habitat were also observed. The
two strains S3 and S4 were identified as Spirulina platensis. It was presented in Table 1. The strain S3 and
S4 was grown in Zarrouk's medium for further analysis for 30 days in light chamber. The growth parameters
optical density cell population and dry weight of S3 and S4 strains were estimated. All the growth
parameters were highest in S3 followed by S4. The effect of temperature and pH determined for the strains
S3 and S4 highest biomass was observed at 30°C of pH 9.5. In this optimum condition the biomass observed
S3 and S4 were found to be 4.6 and 4.2 mg/100 ml. It has been shown by previous workers (Danesi et al.,
2001; Vonshak, 1997) that the optimal growth temperature for S. platensis is between 30°C and 35°C with
40°C definitely being deleterious to this cyanobacterium. In respect to increase in biomass, the best
responses were obtained at 30°C, which agrees with the studies by Danesi et al. (2001).
The rice mill effluent was collected from Parvathy rice mill from Sethiyathopu. The waste water
observed that the colour was pale yellow, the odour was unpleasant and the effluent were turbidity with an
acidic pH (6.09) with low concentration of DO (0.2 – 1.0), BOD (350), COD (550), nitrate (2.5mg), sodium
(100.09 ppm), calcium (50.36 ppm), pottasium (8 ppm), magnesium (43.78 ppm). Moreover the waste
water was rich major minerals like sodium, calcium and magnesium (Table 3). It was stored in cold room
temperature at 4ºC.
Spirulina strains S and S were cultivated in RME broth. Four 250 ml conical flask were taking
3 4
-1
and add 100 ml of rice mill effluent, in each conical flask various Nitrogen (NaNO and KNO 1gL ) and
3 3
-1
Phosphorus (K HPO and KH PO 1gL ) were added separately. In both nitrogen sources high algal
2 4 2 4
biomass was observed in S , NaNO (dry weight 3.80 mg / 100 ml) followed by KNO (dry weight 2.80 mg /
3 3 3
100 ml). In S , NaNO (dry weight 3.73 mg / 100 ml) followed by KNO (dry weight 2.73 mg / 100 ml) was
4 3 3
observed (Table 4). Carvalho et al, 2010 reported that the presence of nitrogen source was necessary to
ensure higher cell mass concentration. Thus justifying the traditional use of nitrate in Spirulina platensis
culture media. Nitrogen deficiency, as visually confirmed by the yellowish colouring of cells observed up
to the 10th day of cultivation. Afterward, the use exponentially increasing flow rates of nitrogen sources
were likely responsible for higher amount of nitrogen provided to the system, for increased pigment
formation and then for restoring of cell dark green colour.
Phosphorus is a major nutrient required for the growth of alga and determines its primary
productivity. Mostert and Grobbelaar, 1981 have indicated the essential role of phosphorus in maintaining
high production rates of microalgae mass cultures. In phosphorus sources high algal biomass was observed
in S , K HPO (dry weight 4.20 mg / 100 ml) followed by KH PO (dry weight 2.73 mg / 100 ml). In S ,
3 2 4 2 4 4
K HPO (dry weight 4.00 mg / 100 ml) followed by KH PO (dry weight 2.50 mg / 100 ml) was observed.
2 4 2 4
The low cost RME media was standardized and compared with routine ZM media composition
shown in Table (5). This investigation was taken up with the basic aim of providing a simple, locally
available, eco-friendly and low cost medium.
Spirulina has a high bicarbonate requirement, which acts not only as a carbon source but helps to
maintain alkaline conditions, and increase the growth of Spirulina. Since laboratory grade sodium
bicarbonate is costly in the Indian context, in RME medium 10 gL-1 compare with ZM media 16.8 gL-1
Therefore, the significant of the RME medium are clearly emphasized, not only as a low-cost alternative
but also as a highly productive input, which can be used profitably by the rural population for large-scale
biomass production of protein-rich Spirulina. In tropical countries, especially developing countries such as
India, emphasis is placed more on the production costs. Therefore the present investigation was aimed
towards the formulation of a new cheaper, low cost RME medium for the growth of cyanobacterium
Spirulina (a rich source of proteins), using locally available rice mill waste water and create eco-friendly
environment. The present study, the above discussed results are related to the formulation of low cost RME
media, and production of Spirulina SCPand reduce the cost of production.
REFERENCE
1.Ciferri. O. 1983. Spirulina the edible microorganism. Microbiol Rev, 47: 551- 578.
2.Cohen, Z., 1997. The chemicals of Spirulina. In: Vonshak, A. (Ed.), Spirulina platensis
3.Danesi, E.D.G., Rangel, C.O., Pelizer, L.H., Carvalho, J.C.M., Sato, S and Moraes, I.O. 2001. Production
of Spirulina platensis under different temperature and urea feeding regimes for chlorophyll attainment. In:
Proceedings of the Eight International Congress on Engineering and Food, 2: 1978-1982.
4.Noorjahan C.M., Sharief D.S., Dawood N. (2005) Biodegradation of dairy effluent, Pollution Research,
Vol. 24, pp. 101-104.
5.Olguin. J, Sonia Galicia, Ofelia Angulo Guerrero and Elizabeth Hernandez (2001). The effect of low light and nitrogen deficiency on the chemical composition of Spirulina sp. (Arothrospira) grown on digested pig
waste, Bioresource Technology, 77: 19-24.
6Pradhan, A. and S.K.Sahu (2004).Process details and effluent characteristics of a Rice mill in the
Sambalpur district of Orissa. J. Ind. Pollut. Cont., 20, 111-124.
7.Vonshak, A., 1997. Spirulina platensis (Arthospira). Physiology, Cell-biology and Biotechnology. Taylor
and Francis. London. ISBN 0-2035-8670-0.
8.Vonshak. A, Abeliorick. A, Boussiba. S, Arad .S and Richmond. A. 1982. Production of Spirulina biomss:
effect of environmental factors and population density, Biomass, 2: 175-185.
Indian Streams Research Journal • Volume 2 Issue 12 • Jan 2013 7
FORMULATION OF COST EFFECTIVE RICE MILL EFFLUENT MEDIUM .........
K.AMALA., M.Sc., M.Phil., Ph.D. Research Scholar in Dept. of Microbiology,
Annamalai University, Chidambaram, 2011-2013.
ADDITIONAL QUALIFICATION:
Medical coding in Hitech medicod Trichy
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