Int.J.Curr.Microbiol.App.

Sci (2015) 4(3): 56-63

ISSN: 2319-7706 Volume 4 Number 3 (2015) pp. 56-63

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Original Research Article

Production Characterization and Aqua Feed Supplementation of Astaxanthin
from Halobacterium salinarium

G.Ramanathan1*, P.Ramalakshmi1, B.Gopperundevi1 and J.Immanuel Suresh2

1
Research Department of Microbiology- V.H.N.Senthilkumaranadar College,
Virudhunagar-626001, Tamilnadu, India
2
Department of Immunology and Microbiology, The American College, Madurai, India
*Corresponding author

ABSTRACT

Halobacterium salinarium is an obligate halophilic archaeon highly adapted to

environments of extremely high salinity. Halobacteria produce large quantities of
Keywords red-orange carotenoids. Carotenoids are a class of natural fat soluble pigments
which produced orange-red colour. Astaxanthin is the main carotenoid extracted
Astaxanthin, from Halobacterium salinarium, which cannot be synthesized by animals and must
Cyprinus carpio, be provided in the diet. It is important to produce protein rich food in an aquatic
Halobacterium system to improve the growth and bioenergetics of an aquatic species. In this study,
salinarium, astaxanthin was used as a supplement for aquafeed and tested against bioenergetics
Live stock of the ornamental fish Cyprinus carpio. The weight, length of the Cyprinus carpio
fed with astaxanthin is increasing with increasing content of halobacterial
astaxanthin supplementation.

Introduction

Halophiles are salt loving organisms that genus Haloterrigena has recently been
grow best at the highest salinities (3 4.5 mol proposed (Dym et al., 1995).
L-1 Nacl) forming dense blooms and
resulting in the red colour of many brines. The first microbiological analysis was
Common species of halobacteria are rod, conducted on several closely related
cocci, or disc shaped, although triangular Halobacterium strains (originally designated
and even square shaped species exist. as H. salinarium, H. halobium, H.
Halobacteria are classified as archea and cutirubrum) isolated in the mid-twentieth
belongs to the family halobacteriaceae. Ten century from salted fish and meat from
genera have been reported, Halobacterium, North America.
Haloarcula, Halococcus, Haloferax,
Halorubrum, Halobaculum, Halobacterium salinarium is an obligate
Natronobacterium, Natranococcus, halophilic archaeon highly adapted to
Natrialba and Natromonas and an eleventh environments of extremely high salinity.
Halobacterium salinarium species primarily

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inhabit thalassohaline salt lakes and solar Astaxanthin has antioxidant potency 1,000
salterns, thereby exhibiting optimal growth times greater than Vitamin E and 10 times
in the presence of NaCl concentrations greater than any known carotenoid. It also
between 2M and saturation. Halobacterium stimulates and improves the immune
salinarum is an aerobe chemoorganotroph response by enhancing immunoglobulin
growing on degradation products of other production in human blood cells.
organisms (Ng et al., 2000).
Astaxanthin cannot be synthesized by
Halobacteria produce large quantities of animals and must be provided in the diet.
red-orange carotenoids. Carotenoid Mammals lack the ability to synthesize
pigments are the most important and astaxanthin or convert dietary astaxanthin
numerous pigments that are found in nature. into vitamin A; unlike -carotene,
astaxanthin has no provitamin A activity in
These compounds soluble in lipids are the these animals (Jyonouchi et al., 1995).
factors that produce yellow-red colour in
plant and animal products. In this group of Aquaculture is broadly defined as the
pigments astaxanthin has important farming of aquatic organisms such as
applications in human and animal food prawns, mollusks, seaweeds, algae in
industries specifically pharmaceuticals and addition to various fishes. In India Mumbai,
cosmetic industries. The Food and Drug Mysore, Bangalore and Kolkata aquarium
Administration of the United States has are made in which ornamental fishes are
permitted it for use in the aquaculture cultured (Shamini and Bhatnagar, 2002).
industry (Golkhoo et al., 2006).
Aquaculture is currently one of the fastest
growing food production systems in the
world. This is emerged as an industry and
now possible to supply protein rich food
throughout the world

Molecular formula: C40H52O4 In this present investigation the astaxanthin

produced by marine bacterium
Common name: 3,3 -dihydroxy- -carotene- Halobacterium salinarium was used as a
4, 4 - dione supplement for aqua feed and tested against
bioenergetics of the ornamental fish
Astaxanthin is the main carotenoid found in Cyprinus carpio.
aquatic animals (Miki, 1991). This red
orange pigment is closely related to other Materials and Methods
well known carotenoids such as -Carotene,
Lutein. Isolation of marine soil sample

In many of the aquatic animals in which it is The marine Halobacterium salinarium was
found, astaxanthin has a number of essential previously isolated by primary selection
biological functions, ranging from process from the marine soil sample
protection against UV light, immune collected from saltpan region of Mukkani,
response, pigmentation and communication Tuticorin District by serial dilution and pour
to reproductive behavior and improved plate technique.
reproduction (Meyer et al., 1993).
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Identification of the Halobacterium Spectrophotometric assay of astaxanthin

salinarium
A separating funnel containing extracted
The pure culture of Halobacterium pigment astaxanthin with acetone solution
salinarium was identified based on their and 0.5 times of petroleum ether as well as
morphology and colony characteristics. The two milliliters of a solution of saturated
organisms were maintained on Halophilic NaCl solution. The petroleum ether phase
agar medium or Zobell marine agar medium was collected after removal of the acetone
and stored at 4°C (May, 2000). layer, which was reextracted with acetone
and the absorbance was read at 471nm.
Pure cultures of Halobacterium salinarium Astaxanthin composition was calculated by
was selected and screened for astaxanthin the following formula, using 1% extinction
pigment production in Halobacterium coefficient = 2100 (Weber, 1990).
salinarium selective medium
ml. of petroleum ether x A471 x 100
Extracted astaxanthin = ---------------------------------------
Optimization of culture conditions (µg/g of bacterial cell) 21 x bacterial dry weight

An attempt was also made to determine the Thin-layer chromatography

optimum culture conditions for
Halobacterium salinarium such as pH, The broth culture medium was filtered and
temperature requirements for their centrifuged at 3000rpm for 5 minutes. Three
maximum pigment production. Five groups of solvents; Group1 Petroleum ether:
different cultivation medium with different Acetone: Diethylamine (10:4:1) Group 2
pH (5, 6, 7, 8 and 9) different temperature Hexane: Acetone (3:1) Group 3 Benzene:
(30°C, 35°C, 40°C, 45°C and 50°C) and Ethyl acetate (1:1) were added to the
NaCl concentrations (5%, 7%, 10%, 15%, extracted astaxanthin. Concentrated
and 20%) were used and extracellular astaxanthin extract was spotted on a TLC
pigment production of the selected strain plates and eluted with a mobile phase of
was recorded. hexane: acetone (3:1) The plate was then
allowed to run approximately three inches
Extraction of astaxanthin pigment above the sample line and then it was
produced by Halobacterium salinarium removed (Lorenz Todd, 1998). The Rf value
can be calculated using the following
Pigment was extracted from the selected formula
strain of Halobacterium salinarium by
centrifugation at 8000rpm for 10mins. The Distance travelled by the sample
precipitate was then resuspended in distilled Rf = -------------------------------------------
water. After a short period, cellular lysis was Distance travelled by the solvent
occurred.
HPLC, NMR and FTIR analysis
Later, the direct extraction of pigment with
acetone was done (Calo, 1995). The pigment The spot obtained from thin layer
obtained was used for the further chromatography was redissolved in acetone
spectrophotometric and chromatographic and was subjected for HPLC, NMR and
analysis. FTIR analysis then the results were
recorded.

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Astaxanthin feed supplementation and coloured colony in the Halobacterium

preparation salinarium selective medium. The red
orange colour colony produced on the
For the present study, the control and halophilic medium indicates that the
experimental feed were formulated by the organism has ability to produce the
following method described by New (1987). astaxanthin. Among the five different media
The experimental feed was prepared by used in the present study, the maximum
using astaxanthin as a source of protein with astaxanthin production was obtained in
8 different concentrations such as 0.4g, 0.8g, Halobacterium medium-1. The optimum
1.2g, 1.6g, 2.0g, 5.0g, 10.0g and control feed condition for the production of astaxanthin
(devoid of astaxanthin). During the culture was 35°C and at the pH 7.
period of 40 days, feeding was done once in
daily. An orange colour spot with Rf value of 0.50
observed through thin layer
Biochemical analysis of feed chromatography. The UV- spectral analysis
showed that the maximum absorption at
Protein is estimated by Biuret method 471nm which indicates the astaxanthin
(Lowry et al., 1951) and carbohydrate is pigment (Fig. 1). The HPLC analysis
estimated by Phenol-Sulphuric acid method showed, the retention time at 3.685, it
(DuBois et al. (1956). Bio energetics also confirmed the astaxanthin pigment (Fig. 2).
estimated by the following formulae: The NMR analysis revealed that the data for
protons present in (C40H52O4) astaxanthin
(Fig. 3). The FTIR analysis revealed that the
Assimilation Assimilation (A) data for the functional groups of the
efficiency (AE) = -------------------------- x 100 astaxanthin pigment (Fig. 4).
Food consumption (C)
Among the eight formulated diets, the
Gross growth efficiency (KI) = P/CX100 protein and carbohydrate content was found
maximum in the astaxanthin supplementary
Net conversion (or)
net growth efficiency (K2) = P/AX100 feed (T7). The protein and carbohydrate
content was found maximum in the
In. Final weight In. Initial weight Cyprinus carpio fed with astaxanthin
Assimilation = ------------------------- x 100 supplementary feed (T7) when compared
efficiency (AE) Experimental period (days) with control. The fish growth characteristics
such as length, weight, assimilation,
Food Dry food consumed metabolism, specific growth rate, gross
conservation ratio (FCR) = ------------------------ growth efficiency, net growth efficiency and
Wet weight gain consumption efficiency in Cyprinus carpio
were found better in the ten gram of
Results and Discussion astaxanthin supplementary diet fed to the
animal than in control (Table 1). In the
The marine soil sample was collected from present study, it was found that the single
saltpan region of Mukkani, Tuiticorin source of astaxanthin enriched diet is found
District, the marine Halobacterium was better FCR (1.79) could be used as
isolated. The isolated culture was identified alternative feed supplement.
as Halobacterium salinarium due to its
capability to produce the red orange

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Table.1 Bioenergetics of Fish Cyprinus carpio after treatment with Experimental Feed

Assimilation efficiency

consumed/Wet weight
(%) SGR=W2-W1/20×

Net growth efficiency

Assimilation A= C-F

Specific growth rate

Food consumed (C)

Metabolism R=A-P

(%) AE=A/C× 100

(%) K2=P/A× 100

Mid body weight

efficiency (%)=

FCR=Dry food
efficiency (%)
Faecal output

Gross growth

Consumption
Initial weight

K1=P/C× 100

C/W/10× 100
W=W2-W1/2
Final weight

Production

(mg) F
Diets

gain
100
T1 5.15 5.45 0.30 0.150 0.36 0.050 0.310 0.010 1.50 66.66 83.33 103.33 24.00 2.18

T2 5.19 5.47 0.28 0.140 0.40 0.090 0.310 0.030 1.40 68.50 70.00 90.32 28.00 2.17

T3 5.23 5.50 0.27 0.135 0.96 0.129 0.831 0.561 1.35 86.56 28.12 32.49 38.50 2.06

T4 5.38 5.93 0.55 0.275 1.06 0.206 0.854 0.304 2.75 80.56 51.88 64.40 43.65 2.09

T5 5.60 6.30 0.70 0.350 1.64 0.236 1.404 0.704 3.50 85.60 42.68 49.85 46.82 2.00

T6 5.67 6.40 0.73 0.365 2.36 0.243 2.117 1.387 3.65 89.70 30.93 34.48 65.56 1.86

T7 6.02 7.60 1.58 0.790 3.45 0.296 3.154 1.574 7.90 92.76 45.79 50.09 48.65 1.79

T8 5.24 6.23 0.99 0.495 1.16 0.120 1.040 0.050 0.19 89.65 85.34 95.19 23.04 1.65

T1-Experimental feed + 0.4g of astaxanthin, T2-Experimental feed + 0.8g of astaxanthin, T3-Experimental feed +
1.2g of astaxanthin, T4-Experimental feed + 1.6g of astaxanthin, T5-Experimental feed + 2g of astaxanthin, T6-
Experimental feed + 5g of astaxanthin, T7-Experimental feed + 10g of astaxanthin, T8-Control feed devoid of
astaxanthin (Trio).

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Fig 1 : UV Spect ral Analysis of Ast axathin Fig 3 : H - NMR Spectral Analysis of Astaxathin

Fig 2 : HPLCAnalysis of Astaxat hin Fig 4: FTIR Analysis of Astaxathin

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The present investigation reveals that the are very applicable in aquaculture
culture condition of Halobacterium production systems. Fish fed with
salinarium significantly enhances high astaxanthin diets outperformed all other
production of astaxanthin in high salt treatments with both superior growth rates
concentration. The feed supplemented with and FCR values. Thus it can be used for
10gms of astaxanthin increases the growth aquaculture feed.
and bioenergetics of the test organism
Cyprinus carpio. The obtained results which Acknowledgements
are similar to the previous study of
astaxanthin production by an extremely The author are thankful to the authorities of
halophilic archea bacteria isolated from high V.H.N.S.N. College, Virudhunagar, Tamil
salt fermented Thai foods (May and Nadu, India for providing required facilities
Paukatong, 2000). to complete this work.

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