Chapter 6
BIOCHEMICAL CHARACTERIZATION
AND PIGMENT PROFILE OF EUGLENA
AND ITS USE AS FEED IN SOME
INDIGENOUS FISHES OF CACHAR
DISTRICT (ASSAM)
BIOCHEMICAL CHARACTERIZATION AND PIGMENT PROFILE OF
EUGLENA AND ITS USE AS FEED IN SOME INDIGENOUS FISHES OF
CACHAR DISTRICT (ASSAM)
6.1 Introduction
The pigments are characteristic of certain algal groups. Four different kinds of pigments that are
usually found in algae are chlorophyll, carotenes, xanthophylls and phycobilins but in Euglena is
devoid of phycobilins pigments. Of the other three kinds, chlorophyll a, chlorophyll b, carotene,
-carotene, zeaxanthin, flovoxanthin, flavicin are common pigment in Euglena.
Chlorophyll and carotenes are fat soluble molecules and are extracted from thylakoid membranes
with the help of organic solvents such as acetone, methanol, etc. but phycobilins and peridinin
are water soluble that can be extracted from algal tissues after the organic solvent extraction of
chlorophyll from those tissues. In some cases, red colouration in the water occurs due to the
increase in presence of characteristic xanthophyll pigment called astaxanthin or euglenorhodone
or hematochrome. Cunningham and Schiff (1986a) found that Euglena contains xanthophyll
pigments diadinoxanthin and diatoxanthin but lutein, fucoxanthin and violaxanthin are not
present. Casper-Lindley and Bjorkman (1998) observed at high light intensities Euglena lacked
xanthophylls pigment. The definition of photosynthetic pigments that cause light energy to turn
into chemical energy in all photosynthetic organisms was first determined by Stokes (1864).
Sorby (1873) classified blue chlorophyll as chlorophyll a, green chlorophyll as chlorophyll b and
orange-yellow as xanthophyll according to the pigment colors. Chlorophyll is a key biochemical
component in the molecular apparatus that is responsible for photosynthesis. Chlorophyll is a
metal-chelate, or a central magnesium ion is bonded to a larger organic molecule called a
porphyrin. The porphyrin molecule is composed tetrapyrrole units joined vinylic groups. The
magnesium ion is the centre of electron transfer during photosynthesis (Fig. 6.1). The content of
chlorophyll a relating to the pigment level is almost the same in all algal groups but chlorophyll
b and c changes(Donkin, 1976; Martin et al., 1991; and Grung et al., 1992). Carotenoids are
naturally occurring fat soluble pigments that are responsible for the different colours of algae
(Ben-Amotz and Fishler, 1998). Carotenoids are usually yellow to red and present in green,
yellow, leafy vegetables and in yellow fruits. They are aliphatic hydrocarbons consisting of
polyisoprene backbone. Pigmentation in Euglenophyta is found to be due to the presence of
carotenoids such as carotene, zeaxanthin, neoxanthin and diadinoxanthin (Goodwin, 1976; Kirk
and Tilney-Bassett, 1978). Rosowski and Parker (1982) found the presence of zeaxanthin, lutein,
violaxanthin and neoxanthin in euglenophyceae. Earlier it was found that secondary carotenoids
such as ketocarotenoids of xanthophylls were associated with chlorophyceae (e.g., green algae),
but then it had been encountered in euglenophyceae. The role of carotene pigments in algae is
not exactly known but it is suggested that they function as a passive light protecting filter and
have the role of accessory pigments transferring energy and oxygen (Lichtenthaller, 1987; Yong
and Lee, 1991 and Bidigare et al., 1993). In Euglena, carotenoids are found to play a major role
in protecting chloroplasts against photosensitized oxidation (Bamji and Krinsky, 1965). Vechetel
et al., (1992) determined that carotene pigments are the most important photosynthetic pigments
and it prevents chlorophyll and thylakoloid membrane from the damage due to photo-oxidation.
Carotenoids contain a conjugated double bond system of the polyene type (C-C=C-C=C). Energy
absorbed by carotenoids are transferred to chlorophyll a for photosynthesis.
Fig. 6.1: Molecular structure of chlorophyll
Various studies have indicated that carotenoids may prevent or inhibit certain types of cancer,
arthrosclerosis, age-related muscular degeneration and other diseases. At sufficiently high
concentrations, carotenoids can protect lipids from peroxidative damage (Burton, 1984).
Carotenoids have antiproliferative effect on various cancer cell lines; lycopene has been shown
- Carotene has been
shown to inhibit the expression of antiapoptotic protein Bcl-2 in cancer cells, thus reducing the
growth of cancer cells (Karas, 2000). Paramylum is the characteristic carbohydrate reserve of the
Euglenophyta (Braas and Stone, 1968) which is similar to starch. It accumulates when Euglena
are grown on an organotrophic medium in the dark and is consumed either in the dark when cells
receive or at the end of the exponential growth phase (Freyssinet et al., 1972) or when cells are
transferred to the light (Dwyer and Smillie, 1970; Dwyer and Smillie, 1971; Freyssinet 1972;
Schwartzbach et al.,1975). The chloroplasts found in Euglena contain chlorophyll which aid in
the synthesis of carbohydrates is stored as starch granules and paramylon. In Euglena paramylon
is made in the pyrenoids. The eugenoids have chlorophylls a and b and they store their
photosynthate in an unusual form called paramylon starch, a B-1, 3 polymer of glucose. The
paramylon is stored in rod like bodies throughout the cytoplasm. These are called paramylon
bodies and are often visible as colorless or white rigid rods (Calvayrac, 1981). Protein or amino
acids are the by-products of an algal process for the production of other fine chemicals, or with
appropriate genetic enhancement, microalgae could produce desirable amino acids in sufficiently
high concentrations (Borowitzka, 1988). The high protein content of various algae species is one
of the main reasons to consider them as an unconventional source of protein (Soletto et al.,
2005). Euglena is an organism with a number of interesting characteristics. It has three, rather
than two membranes surrounding its chloroplasts (Gibbs, 1978) which have implications for the
targeting of nuclear-encoded chloroplast proteins. It is a popular flagellated laboratory
microorganism found in freshwater environments (Buetow et al., 1982). It represents one of the
simplest and earliest derived eukaryotic cells. The production of variety of extracellular
substances plays an important role in growth, physiology and ecosystems of algae. Extracellular
products which are liberated from Euglena contain lot of nitrogenous substances.
Producer
Product group
Application
Euglena ( - carotene)
Carotenoids
Pigments, cosmetics,
Pro-vitamins
Euglena gracilis
Vitamin C and vitamin E
Nutrition
-tocopherol, ascorbic acid)
Algal blooms are often disastrous in aquatic bodies and particularly in fish ponds due to the
addition of fertilizers it causes eutrophication and alter the quality of water which results into
fish mortality (Padmavathi, 2007). Most of the works deals with the physico-chemical
parameters operating in a particular water body while some workers have discussed about the
distribution of unicellular and colonial organisms. In 2000, Hosmani and Vasanth work on the
biochemical aspects of water pollution in two lakes of Mysore city. Rahman et al., (2007)
studied on Euglena bloom and its impact on fish growth. They correlated the water quality in the
bloomed ponds with fish growth and found that bloom had a negative effect on fish growth.
Though they did not analyze the gut contents of the fishes inhabiting the same ponds. Most
researches on fish growth are based on impact of water quality on fish growth (Vas, 2006;
Sugunan et al., 2006)
So far biochemical aspects of algae have received less attention in natural habitats of freshwater
ecosystems (Gatenby et al., 2003). The present chapter embodies the results of investigation on
the biochemical potential and pigment profile of Euglena tuba. Also included in this chapter
some informations on intake of Euglena tuba by the indigenous fishes to explore its innate
potential as fish feed.
6.2 Methodology
For the biochemical characterization and pigment profile analysis, all the 16 ponds were
selected and algal samples were collected bimonthly from July-2009 to May- 2010. Euglena
intake information was obtained by analyzing the guts of some indigenous fish variety.
Informations pertaining to the fish kill was obtained by interaction with the local people through
a structured questionnaire. The detail methodology pertaining to the work described in this
chapter has already been discussed in Chapter 3.
6.3 Results and Discussion
6.3.1 Pigment profile of Euglena tuba
Pigment concentration and biochemical properties are largely dependent upon the type of algae.
Euglena that grows at different light intensities show remarkable changes in their chemical
composition, pigment content and photosynthetic activity (Guschina and Harwood, 2005).
Chlorophyll a, b and carotenoids are fat soluble pigment. Table 6.1 shows characteristics of
different photosynthetic pigments. In general, carotenoid was found highest in almost all the
ponds (Fig.6.2 - 6.17) except Madhuraghat, Dudhpatil, Udarband, Barjalenga and Sonai.
Chlorophyll b was always lower than other pigments. In Arkatipur, Chlorophyll a (7.18µg/ml)
and b (0.85µg/ml) were highest in March. Both chlorophyll and carotenoids were low in July.
The chlorophyll a is an index of water quality and phytoplankton biomass (James and Head,
1972; Papista et al., 2002; Desortova, 1981; Canfield et al., 1985; Voros and Padisak, 1991).
Carotenoid gradually increased from the month of July then decreased with a fall in May. While
chlorophyll a was found to be more or less stable. During March and May, in Baskandi
carotenoid showed heavy fluctuation with its lowest concentration in January (2.5µg/ml) when
chlorophyll b was highest (0.9µg/ml). In Karikandi, carotenoid was found to increase initially
from the month of July to November with a sudden fall in January then gradually increased
towards May. A decrease in chlorophyll a concentration from 6.0µg/ml to 3.5µg/ml was noticed
in the month of May. In Machpara, chlorophyll a and carotenoid shown a similar trend from July
to May while chlorophyll b has a fall from November to May. Carotenoid concentration fell
down to its lowest point in March (2.3µg/ml) then again increased in May (8.6 µg/ml) in
Kashipur while chlorophyll a did not differ much in its concentration. In Bagpur, lowest
carotenoid was observed in July (2.5 µg/ml) when both chlorophyll a and b were highest (9.3
µg/ml and 0.5 µg/ml). Chlorophyll b was greatly fluctuating in Madhuramukh pond with its
highest value (0.5µg/ml) in May when carotenoid was lowest (3.3µg/ml). In Madhuragha,t
chlorophyll a was higher than the carotenoid from the month of November. Increase in
chlorophyll a (9.5 µg/ml) is followed by decrease in carotenoid in March (2.0µg/ml).
Chlorophyll b gradually increased from September (0.1 µg/ml) to March (0.3µg/ml) then fell
down in May (0.1µg/ml). Increases in chlorophyll a concentration in the water and pH is related
to Euglena density whereas oxygen concentration changes are related to the changes in the
density of euglenophytes (Pereira et al., 2001). Chlorophyll b had its peak in January in
Dudhpatil. In this pond chlorophyll a was always higher than the carotenoid. The high level of
antioxidant property in Euglena species is contributed by the fact that it contains carotenoid
pigment. Carotenoid pigment is a potent radical scavenger and singlet oxygen quencher
(Gouveia, 2008). Carotenoids are known to posses antioxidative properties (Di Mascio et al.,
1991, Kobayashi et al., 1997, Fang et al., 2002). The antioxidant activity of carotenoids arises
primarily as a consequence of the ability of the conjugated double-bonded structure with
delocalize electrons (Mortensen, 2001). This is primarily responsible for the excellent ability of
-carotene to physically quench singlet oxygen without degradation and for the chemical
-carotene with free radicals such as the peroxyl, hydroxyl and superoxide radicals.
In Durgabari, carotenoid was lowest in September (1.21µg/ml) and highest in May (10.87µg/ml)
when chlorophyll a and b reached it’s lowest. Euglenophytes (Euglena) made up more
chlorophyll a than diatoms, chlorophytes and especially cyanobacteria (Reynolds, 1984, Pereira
et al., 2001). Carotenoid was found to be more or less stable from July (2.11µg/ml) to November
(2.05µg/ml) then decrease then increased again in Udarband. In Silcoorie, chlorophyll b was
found to be lowest in September (0.09µg/ml) while highest in March (0.45µg/ml) which
decrearses again in May (0.32µg/ml). Generally, higher chlorophyll a concentration translate
into higher individual cell counts and biomass of phytoplankton, though not always, as not all
algal cells produce equal amounts of chlorophyll a (Felip and Catalan, 2000). In Dargakona,
highest chlorophyll a (11.97µg/ml) and b (0.67µg/ml) was followed by lowest carotenoid
(0.99µg/ml) in March. The pigment chlorophyll a, b and carotenoid were found to be fluctuating
from July to May in Irangmara pond. Chlorophyll a (9.54µg/ml) and b (0.32µg/ml) were highest
in Jaunary when carotenoid (3.12µg/ml) was lowest in Barjalenga. In Sonai, chlorophyll a
increased gradually from the month of July than decreased in May. Cunningham and Schiff
(1986) mentioned that the amount of chlorophyll b was relatively lesser than chlorophyll a.
6.3.2. Biochemical characterization of Euglena tuba
In general Carbohydrate concentration was found to be more than the protein content of the
species. Euglena gracilis is reported to have high protein content Chae et al. (2006). Algal cell
mainly made up of proteins, carbohydrates, fats and nucleic acids in varying proportions but their
percentages can vary with the type of algae, some types of algae are made up of up to 40% fatty
acids based on their overall mass. It is this fatty acid that can be extracted and used as biofuel
(Gross, 2009). Fig.(6.18 -6.33) show variation of carbohydrate and protein concentrations in
each pond ecosystem. In Arkatipur Carbohydrate was higher in all the months except March
when protein (102.94µg/ml) was higher than the carbohydrate(97.23µg/ml) concentration. In
Baskandi, Karikandi, Machpara, Kashipur, Bagpur, Madhuramukh, Madhuraghat, Dudhpatil,
Udharband, Silcoorie, Irangmara and Sonai carbohydrate concentration was always higher than
the protein value. While in other ponds such as Durgabari, Dargakona and Barjalenga
fluctuations in carbohydrate and protein concentrations were observed in between the months.
Pigment concentrations most essentially the chlorophyll a concentration of a species which is a
measure of biomass is associated with all other biochemical parameters. Statistical analysis of
the pigments and biochemical properties of Euglena tuba (Table 6.2) showed a significant
negative correlation (p
a and Carotenoid pigment. Higher Chlorophyll
a pigment was associated with higher carbohydrate and protein value (p
Euglena tuba was found to be rich in macro and micronutrient concentrations (Table 6.3-6.4).
Among three major macronutrients nitrogen showed high value (2.0%) followed by phosphorus
(0.32%) and least value was observed in potassium (0.3%). For micronutrients high value was
found in manganese (1178 mg/kg) which was much more as compared with other nutrient
followed by iron (89.6 mg/kg). Zinc concentration was recorded as 75.9 mg/kg and least value
was found for copper (3.5 mg/kg).
6.3.3 Euglena intake by indigenous fishes
Euglena tuba is a dominant unicellular alga in the fishery ponds of Cachar district. Euglena sp.
are known to reduce significantly other groups of algae (Hosmani, 1988). In our study dissolved
oxygen content never reached below 5 mg/l (Chapter- 4) level providing a favorable
environment for the fish survival. Blooms are known to deplete oxygen level in the aquatic body
causing huge fish mortality but in our study Euglena tuba blooms were not found to be harmful
for the fish growth rather they found to provide ample amount of oxygen to the environment by
the process of photosynthesis. This finding also derive support from earlier work by Brunson et
al. (1994). The district has not yet been reported to have any fish kill event due to such bloom.
Though more than 75% of freshwater fishes feed on plankton in their different stages of life
cycle (Jafri et al., 1999) and despite rich availability of the Euglena tuba in fishery ponds
application of the alga as fish feed is uncertain. Lu et al. (2004) also reported that larval tilapia
Oreochromis niloticus ingested significantly less Euglena gracilis but the ingestion was higher
than the Chlorella vulgaris. Fishes include (Plate 6.1) Darkina (Esomus danricus), Jati puti (
Puntius
sophore),
Chepta
puti
(
Puntius
conchonius),
Puta
(Puntius
sarana),
Japani(Cyprinus carpio), Goroi(Chana punctatus), Moka (Amblypharyngodon mola), Ghoria
(Labeo goria), Rohu (Labeo rohita), Puti(Puntius ticto) and Chandhowa (Chanda nama ). The
gut contents of the fishes from the heavily bloomed ponds were analyzed (Plate 6.2) and a very
low number of Euglena could be encountered in these 11 indigenous fish varieties. However Das
et al. (2009) mentioned that intake of Euglena viridis powder in Rohu increases its immunity and
makes Labeo rohita more resistant to Aeromonas hydrophila. Despite the huge abundance of
Euglena tuba in the studied pond ecosystems, food choice of the fishes might be another factor
for reducing the intake of the particular alga in their diet. Irrespective of the food selection and
consumption of the fishes Euglena tuba was found to have rich carbohydrate and protein content
which might have a potential to increase the food value of the same. High nutritive value of
Euglena tuba opens a new area where a future prospect occurs in formulation of the fish feed by
blending Euglena tuba powder according to the fish food habit that may increase the food
selection and consumption rate of the fishes which would help in high and healthier growth rate
of the fishes on commercial basis.
6.4 Conclusion
In general, for all the ponds carotenoid concentration was higher than the chlorophyll a and
chlorophyll b pigments except Madhuraghat, Dudhpatil, Udharband, Barjalenga and Sonai with
prominent bimonthly pigment fluctuations. Carotenoid pigment was rich and found to be
dominant over the chlorophyll a and b pigments and was found to have significant negative
correlation (p
with chlorophyll a. Higher algal biomass in terms of chlorophyll a was
associated with higher carbohydrate and protein value (p
Higher concentrations of macro
and micronutrients in the species may have attributed to higher amount of proteins and
carbohydrate metabolism. Thus Euglena tuba with its well balanced nutrients may pave a new
way towards the fish food formulations which would ultimately increase the commercial output
of the fishery ponds.
Table 6.1: Characteristics of photosynthetic pigments
Pigment
Color
Soluble in
Chlorophyll a
Bluish green
Fat soluble
Chlorophyll b
Yellowish green
Fat soluble
Carotene
Orange
Fat soluble
Table 6.2: Correlation between pigments and biochemical properties of Euglena tuba.
Correlations
Chlorophylla
Chlorophyllb
Carotenoid
Protein
Carbohydrate
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Pearson Correlation
Chlorophylla
1
Sig. (2-tailed)
N
Pearson Correlation
Sig. (2-tailed)
N
Chlorophyllb
Carotenoid
.384**
-.494**
.000
.000
96
.384**
.000
96
96
1
-.494**
.000
96
.771**
.071
.493
96
.162
.000
96
.726**
.000
96
Protein
Carbohydrate
.771**
.726**
.000
.000
96
.071
.493
96
96
.162
.115
96
96
.184
.072
96
96
-.525**
-.525**
.000
96
1
-.458**
.000
96
.633**
.115
96
.184
.000
96
-.458**
96
.633**
.000
96
1
.072
96
.000
96
.000
96
96
1
**. Correlation is significant at the 0.01 level (2-tailed).
Table 6.3: Macro-nutrient content in Euglena tuba
Nutrient
Percentage (%)
Total Nitrogen (N)
2.07
Total Phosphorus (P)
0.317
Total Potassium (K)
0.30
Table 6.4: Micronutrient concentration of Euglena tuba
Trace metal
Iron (Fe)
Manganese (Mn)
Zinc (Zn)
Cupper (Cu)
Concentration (mg/kg)
89.6
1178.4
75.9
3.5
96
Fig. 6.2: Variation of photosynthetic pigments of Euglena tuba in Arkatipur pond
Fig. 6.3: Variation of photosynthetic pigments of Euglena tuba in Baskandi pond
Fig. 6.4: Variation of photosynthetic pigments of Euglena tuba in Karikandi pond
Fig. 6.5: Variation of photosynthetic pigments of Euglena tuba in Machpara pond
Fig. 6.6: Variation of photosynthetic pigments of Euglena tuba in Kashipur pond
Fig. 6.7: Variation of photosynthetic pigments of Euglena tuba in Bagpur pond
Fig. 6.8: Variation of photosynthetic pigments of Euglena tuba in Madhuramukh pond
Fig. 6.9: Variation of photosynthetic pigments of Euglena tuba in Madhuraghat pond
Fig. 6.10: Variation of photosynthetic pigments of Euglena tuba in Dudhpatil pond
Fig. 6.11: Variation of photosynthetic pigments of Euglena tuba in Durgabari pond
Fig. 6.12: Variation of photosynthetic pigments of Euglena tuba in Udarband pond
Fig. 6.13: Variation of photosynthetic pigments of Euglena tuba in Silcoorie pond
Fig. 6.14: Variation of photosynthetic pigments of Euglena tuba in Dargakona pond
Fig. 6.15: Variation of photosynthetic pigments of Euglena tuba in Irangmara pond
Fig. 6.16: Variation of photosynthetic pigments of Euglena tuba in Barjalenga pond
Fig. 6.17: Variation of photosynthetic pigments of Euglena tuba in Sonai pond
Fig. 6.18: Variation of biochemical parameters in Arkatipur pond
Fig. 6.19: Variation of biochemical parameters in Baskandi pond
Fig. 6.20: Variation of biochemical parameters in Karikandi pond
Fig. 6.21: Variation of biochemical parameters in Machpara pond
Fig. 6.22: Variation of biochemical parameters in Kashipur pond
Fig. 6.23: Variation of biochemical parameters in Bagpur pond
Fig. 6.24: Variation of biochemical parameters in Madhuramukh pond
Fig. 6.25: Variation of biochemical parameters in Madhuraghat pond
Fig. 6.26: Variation of biochemical parameters in Dudhpatil pond
Fig. 6.27: Variation of biochemical parameters in Durgabari pond
Fig.6.28: Variation of biochemical parameters in Udarband pond
Fig.6.29: Variation of biochemical parameters in Silcoorie pond
Fig.6.30: Variation of biochemical parameters in Dargakona pond
Fig. 6.31: Variation of biochemical parameters in Irangmara pond
Fig.6.32: Variation of biochemical parameters in Barjalenga pond
Fig. 6.33: Variation of biochemical parameters in Sonai pond
Plate 6.1: Some of the indigenous fish varieties (whose gut analysed) from different ponds
Plate 6.2: Optical micrographs of Euglena tuba obtained from guts of fishes