characterization of carotenoids in algae collected...

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43 �

CHAPTER 3

CHARACTERIZATION OF CAROTENOIDS IN

ALGAE COLLECTED FROM NATURAL SOURCE

3.1 Collection of algal sample

North Eastern region, more particularly Assam is known for its rich aquatic

biodiversity. Algal bloom is the most common natural phenomenon in warmer

shallow and eutrophic water bodies in North Eastern part of India. A few species

of algae are generally floats on the surface of the water and we are interested to

study their carotenoid content. Hence the upper floating layer was collected for

the study. The algal scum were collected from different fishery ponds in the same

season for three consecutive years (2009, 2010, and 2011). In our study, twenty

different fishery ponds were selected randomly from the districts of Kamrup,

Morigaon and Darrang of the state of Assam (Figure 3.1). Algae collected from

the different ponds were given sample identity from AS1 to AS20 (AS stands for

algal sample) corresponding to the each of the twenty selected ponds. The algal

samples were carefully collected in screw cap bottles having proper labels and

markings along with pond water and carried to the culture laboratory for further

study. The pH of the water bodies were also recorded while collecting the

samples (Table 3.12). Many algal species are known to grow better at a

particular pH. So to culture the collected sample maintenance of its pH of the

natural habitat is important. In the laboratory the collected algal biomass was

transferred to a beaker and then stirred vigorously by adding distilled water with

a glass rod and allowed to settle down. Usually the impurities, which float on the

surface of the water, were decanted off and the algal samples were further filtered

and preserved for the present study. This initial purification was followed in each

of the samples collected from the natural ponds.

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44 �

Figure 3.1: Algae samples collected from different natural habitats.

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45 �

3.2a Extractability of carotenoids from algae in different solvent systems

Extractions of carotenoid were tried by different procedures viz grinding

followed by vortexing with glass beads and grinding followed by homogenization

using different solvents and mixture of solvents. Each extraction was carried out

with 1g of the dried algae. 20 mL of each solvent in at least four equal amounts

were used for extraction. The extraction was continued till 20 mL of the extract

was colorless. This had to be repeated to ensure complete extraction of

carotenoids from the algal sample. The total carotenoid present in the extract was

measured by UV-visible spectrophotometer (Shimadzu Model UV- 1601) and

estimated. Three different processes were attempted to have better extractability

of carotenoids from the algal biomass.

In the first process 1g of the weighed algae was grinded with anhydrous sodium

sulphate in a porcelain mortar with a glass pestle and then vortexed with glass

beads using different solvents such as hexane, methanol, acetone,

dichloromethane (DCM), chloroform, ethyl acetate, ethanol, diethyl ether,

dimethyl sulfoxide (DMSO), tolune, isopropanol, n-butanol, heptane, acetonitrile

and tetrahydrofuran (THF). In this set of experiment the maximum amount of

carotenoid was extracted with hexane and the least extraction was found with

ethanol (Figure 3.2a). In case of ethanol, isopropanol and n-butanol, the color of

the residues were found to be red which indicates the incomplete extraction of

carotenoid in these solvents.

In the second process the extraction was carried out after grinding with

anhydrous sodium sulphate in a porcelain mortar and then homogenizing of the

algal samples with hexane, DCM, diethylether, methanol, ethylacetate, THF,

acetone, ethanol, isopropanol and butanol in a glass homogenizer. Other solvents

used in the first set were not included in this set as their extractability was poor.

Although the extraction of carotenoid with ethanol, isopropanol, and n-butanol

was poor, yet they were used in this set considering their aqueous nature. The

amounts of carotenoids extracted in different solvents in set 2 are shown in

(Figure 3.2b).

In the third set, extraction of carotenoids was attempted with mixtures of solvents

containing at least one aqueous solvent in a glass homogenizer. The various

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46 �

solvents used in the mixtures were DEE, ethanol, hexane, acetone, THF, DCM,

ethylacetate, isopropanol, n-butanol and methanol. In the mixtures 30% of

aqueous solvents were used. While using hexane-ethanol/methanol mixtures two

layers were formed in the homogenized extract indicating the immiscibility of the

mixture, for which these mixtures were not used further. In case of hexane- n-

butanol mixture single layer was formed in the homogenized extract. Of the

different mixtures of solvents used, the amount of carotenoids extracted in DCM:

methanol (7:3 v/v) mixture was found to be 19.903 mg/g and in case of acetone:

methanol (7:3 v/v) mixture the total carotenoids extracted was 18.384 mg/g of the

dry algal mass. The extractability of carotenoids in different solvent mixtures is

shown in Figure 3.2c. The results shows that the amount of total carotenoids

extracted with acetone-methanol/ethanol mixture were almost the same as that

extracted with acetone.��

Figure 3.2a: Extraction of carotenoid in different solvents using glass beads.

27

.73

1

16

.75

14.5

38

20.3

85

12.3

85 16.6

73

5.7

71

19

.083

12.3

08

12

.23

1

8.2

69 11 11.8

65

13

.76

5

15.1

15

0

5

10

15

20

25

30

Am

ount

of

caro

teno

id i

n m

g

solvent

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47 �

Figure 3.2b: Extraction of carotenoid in different solvents using a glass

homogenizer

Figure 3.2c: Extraction of carotenoids in different solvent mixtures using a glass

homogenizer

28

.154

25

.80

1

24

.65

4

17.6

53

15.6

73

22

18.1

53

13.3

07

8.6

73

9.9

23

�

�

��

��

��

��

��

Am

ount

of

tota

l ca

rote

noid

in m

g

��

15.2

3

15

.03

8

16

.28

8

17

.5

14.2

59

13

.5

19

.90

3

18.3

84

�

�

��

��

��

��

Am

ount

of

tota

l ca

rote

noid

in m

g

Solvents

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48 �

3.2b Extraction and estimation of total carotenoids in different algal samples

The extraction of carotenoids was carried out under yellow light of the

laboratory. The algae collected from the natural habitat were freeze dried. Around

one gram of the dried algal mass was grinded well with anhydrous sodium

sulphate in a porcelain mortar and then homogenized by using a glass

homogenizer connected to a motor. The cells were homogenized in the

homogenizer with acetone solvent (containing 0.01% BHT in ethanol) for five

minutes and collected in a round bottomed flask after decanting and filtering. The

flask was covered with black cloth to protect from light. The extraction was

repeated until the extract became colorless. The amount of total carotenoid in the

extract was estimated using the procedure of Britton et al. (1995) taking the help

of Shimadzu Spectrophotometer. The UV –Visible spectra (� max) of the algal

extract were also recorded and compared with that of the standard literature

(Britton et al. 1995). The experiments were repeated for three times for each of

the algal sample and the total carotenoids in the different algal samples were

estimated in this way (Table 3.1)

The amount of total carotenoids in the algal extracts were estimated using the

formula as detailed below

A x Y

Total carotenoid (X) = x 106 �g

E1%

1cm x 100

OR

X (�g)

X (�g/g) =

Weight of the sample (g)

Where

X = Weight or concentration of the carotenoids

Y = Total volume ((mL) of the carotenoids extract

A = Absorbance (optical density) at a specified wave length �max

E1%

1cm = Absorption coefficient of the carotenoids in the solvent used.

For mixture carotenoids E1%

cm value was taken as 2500.

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49 �

For each of the algal sample three extractions were carried out and the mean has

been reported in Table 3.1

Table 3.1: Estimated total carotenoids in different algal samples

Sample ID Total carotenoid

(mg/g of dry weight)

�max value

( in nm)

Major carotenoid

present

AS1 6.33 ± 0.31 473 Lycopene

AS2 9.26 ± 0.22 478 Astaxanthin

AS3 4.45 ± 0.21 473 Lycopene

AS4 10.24 ± 0.23 473 Lycopene

AS5 4.60 ± 0.15 473 Lycopene,

AS6 8.97 ± 0.20 473 Lycopene

AS7 11.57 ± 0.34 473 Lycopene

AS8 8.60 ± 0.35 473 Lycopene

AS9 6.64 ± 0.42 473 Lycopene

AS10 23.85 ± 0.36 473 Lycopene


AS12 17.79 ± 0.50 473 Lycopene


AS14 9.68 ± 0.41 473 Lycopene

AS15 6.39 ± 0.50 456 Zeaxanthin

AS16 23.12 ± 0.41 473 Lycopene

AS17 11.60 ± 0.25 473 Lycopene

AS18 9.23 ± 0.51 473 Lycopene

AS19 11.42 ± 0.24 478 Astaxanthin

AS20 11.7 ± 0.35 473 Lycopene

The results are shown as mean ±SD of three experiments

�

The UV Visible spectrum

Figure 3.3: UV-visible spectrum

and AS20 (D) showing lycopene peak at

algal sample AS15 (E) showing zeaxanthin peak

spectrum of algal sample AS11 (F) showing astaxanthin peak a

The UV Visible spectrum of some crude algal samples are shown below

visible spectrum of algal sample AS10 (A), AS12 (B), AS14 (C)

and AS20 (D) showing lycopene peak at �max 473 nm, UV-visible spectrum

algal sample AS15 (E) showing zeaxanthin peak � max 456 nm, and UV

of algal sample AS11 (F) showing astaxanthin peak at � max 478 nm.

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50

of some crude algal samples are shown below

of algal sample AS10 (A), AS12 (B), AS14 (C)

visible spectrum of

456 nm, and UV-visible

478 nm.

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51 �

3.3 Saponification of algal extract

Saponification of algal extract was carried out with Ambersep 900 OH procedure

and methanolic KOH procedure, as mentioned in section 2.3.1 and 2.3.2 of

Chapter 2. The saponified extract was then collected in hexane and the UV

visible spectra were recorded.

(A) (B)

Figure 3.4: Nonsaponified extract (A) of an algal sample, showing the

presence of chlorophyll and carotenoid mixture and saponified extract (B)

showing the presence of carotenoid only.

3.4 Separation and identification of individual carotenoid by TLC

At the preliminary stage, the carotenoids were separated by TLC using Silica Gel

G as the adsorbent. The mobile phase chosen for the best separation of most of

the algal extract was 20% acetone in hexane. Each fraction of the carotenoids was

identified by co-chromatography with the standard carotenoid compounds. The

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52 �

Rf values (defined as the ratio of the distance travelled by the sample to the

distance travelled by the solvent front) of different components were recorded.

Each band was extruded and then extracted with dichloromethane or with hexane

and the visible spectra were recorded. The TLC separated different fractions of

few algal samples with their respective Rf values are shown in Table 3.2.

Table 3.2: TLC separated fractions of some algal samples with their

corresponding Rf values

Algal

sample

Fractions Rf

values

Cochromatography

with standards

Visible

spectrum

Identification

AS10 F1

0.88 lycopene 434, 474,

502

lycopene

F2 0.25 zeaxanthin 421,

456,474

zeaxanthin

AS11 F1 0.88 lycopene 434, 474,

502

lycopene

F2 0.47 astaxanthin 478 astaxanthin

F3 0.32 lutein 421, 444,

474

Lutein

AS15 F1 0.88 lycopene 434, 474,

502

lycopene

F2 0.25 zeaxanthin 421, 456,

474

zeaxanthin

F3 0.32 lutein 421, 444,

474

lutein

AS20 F1 0.88 lycopene 434, 474,

502

lycopene

F2

0.32 lutein 421, 444,

474

lutein

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53 �

3.5 Separation and identification of individual carotenoids by HPLC

The individual carotenoids were separated and identified by using HPLC. The

saponified extract of the algal sample was evaporated to dryness and then

dissolved in a known volume of mobile phase of the HPLC system. 20 �L of the

extract was injected onto the column of HPLC through the injector loop. In the

HPLC column, the individual carotenoids got separated and the diode array

detector recorded and showed their corresponding UV visible spectra. The HPLC

separated carotenoids were identified by comparing the retention times and the

UV visible spectra with that of the authentic standard carotenoids.

3.6 Preparation of standard curve of lycopene, lutein, astaxanthin and

zeaxanthin

A known amount of standard carotenoid was dissolved in a HPLC solvent. 20�L

of this solution was injected in to the HPLC column. In the HPLC chromatogram

the carotenoid shows a peak at a particular retention time. The diode array

detector recorded the area of the corresponding peak. For each of the standard

carotenoid, HPLC chromatogram was recorded with three different

concentrations. On plotting the peak areas against the different concentrations of

the carotenoid, a linear graph was obtained showing the relationships of the

concentrations of the carotenoids and their corresponding areas. This is known as

standard curve of a particular carotenoid. From the standard curves the

concentrations of the desired carotenoids were estimated by injecting a known

amount of a known carotenoid.

3.6.1 Preparation of standard curve of lycopene

By injecting 18.1, 36.7, 75.9 ng of standard lycopene onto a reversed phase

HPLC system a standard curve was obtained (Table 3.3 & Figure 3.5).�

Calibration curve of the standard lycopene were drawn by plotting concentration

in ng vs peak area. A good linear relationship between the concentration and peak

area of the standard lycopene was observed. The linear regression equation of the

calibration curve of lycopene was found to be Y= 15287X-13259 with R2=0.999

(where Y = peak area X= concentration of lycopene). The absolute amount

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54 �

lycopene (in ng) present in the algal extract was calculated from the calibration

curves of lycopene with regression 0.999.

Table 3.3: Estimated amount of standard lycopene and corresponding peak area

from HPLC chromatogram

y = 15287x - 13259R² = 0.999

0

200000

400000

600000

800000

1000000

1200000

1400000

0 10 20 30 40 50 60 70 80

Peak

Are

a

Amount in ng

LYCOPENEE

Figure 3.5: Standard curve of lycopene showing linear relationship between the

concentration and peak area of the standard lycopene.

3.6.2 Preparation of standard curve of lutein

By injecting 12.6, 27.4 and 53.1 ng of standard lutein onto a reversed phase

HPLC system a standard curve was obtained (Table 3.4 & Figure 3.6). The

linear regression equation of the calibration curve of lutein was found to be Y=

15251X + 5158 with R2

= 0.999 (where Y = peak area X = concentration of

lutein). The absolute amount lutein (in ng) present in the algal extract was

calculated from the calibration curves of lutein with regression 0.999.

Estimated lycopene

in ng

Area obtained from HPLC

chromatogram

18.1 255000

36.7 534508

75.9 1155402

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55 �

Table 3.4: Estimated amount of standard lutein and peak area from HPLC

chromatogram

Estimated lutein

in ng


chromatogram

12.6 202284

27.4 425892

53.1 812362

y = 15251x + 5158.

R² = 0.999

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 10 20 30 40 50 60

Peak

Are

a

Amount in ng

LUTEIN

Figure 3.6: Standard curve of lutein

3.6.3 Preparation of standard curve of astaxanthin

By injecting 28.5, 54.6 and 109.5 ng of standard astaxanthin onto a reversed

phase HPLC system a standard curve was obtained (Table 3.5 & Figure 3.7).

The linear regression equation of the calibration curve of astaxanthin was found

to be Y= 67293X-14954 with R2=0.999 (where Y = peak area X= concentration

of astaxanthin. The absolute amount astaxanthin (in ng) present in the algal

extract was calculated from the calibration curves of astaxanthin with regression

0.999.

Table 3.5: Estimated amount of standard astaxanthin and peak area from HPLC

chromatogram

Estimated astaxanthin

in ng


chromatogram

28.5 1946824

54.6 3564587

109.5 7382766

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56 �

Figure 3.7: Standard curve of astaxanthin

3.6.4 Preparation of standard curve of zeaxanthin

By injecting 21.4, 43.2 and 86.5 ng of standard zeaxanthin onto a reversed phase

HPLC system a standard curve was obtained (Table 3.6 & Figure 3.8). The

linear regression equation of the calibration curve of zeaxanthin was found to be

Y= 9442X+3990 with R2=0.999 (where Y = peak area X= concentration of

zeaxanthin. The absolute amount zeaxanthin (in ng) present in the algal extract

was calculated from the calibration curves of zeaxanthin with regression 0.999.

Table 3.6: Estimated amount of standard zeaxanthin and area from HPLC

chromatogram

Estimated Zeaxanthin

in ng


chromatogram

21.4 204332

43.2 422485

86.5 815924

y = 67293x - 14954

R² = 0.999

0

1000000

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 20 40 60 80 100 120

Pea

k a

rea

Amount in ng

Astaxanthin

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57 �

Figure 3.8: Standard curve of zeaxanthin

3.7 Characterization of different carotenoids in the collected algal samples

The individual carotenoids present in the different algal samples were separated

and characterized using HPLC. The saponified algal extracts were evaporated to

dryness and then dissolved in a known volume of solvent which was used in

HPLC system as the mobile phase. 20 �L of the solution was injected into the

HPLC column, where the individual carotenoids were separated and the diode

array detector recorded their corresponding UV Visible spectrum. The

carotenoids were identified by comparing the retention times and the UV visible

spectra of that of the standard carotenoids. All the carotenoids were monitored at

450 nm with UV-visible detector (Shimadzu 1601, Japan). The peak identities

and �max values of these compounds were confirmed by their retention times and

characteristic spectrum of standard chromatograms, recorded with a Shimadzu

model LC-10 ATVP series equipped with SPD-10 ATVP detector. They were

quantified from their peak areas in relation to the respective reference standard

curves. The major carotenoids identified in the different algal samples are listed

in Table 3.7.

y = 9442X + 3990.

R² = 0.999

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

0 20 40 60 80 100

Pea

k a

rea

Amount in ng

Zeaxanthin

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58 �

Table 3.7: The major carotenoids identified in different algal samples

(the highest amount in bold letter)

Sample ID Major carotenoids

AS1 Lycopene, Lutein

AS2 Astaxanthin, Lycopene


AS4 Lycopene, Astaxanthin





AS9 Lycopene, Zeaxanthin


AS11 Astaxanthin, Lycopene, Lutein



AS14 Lycopene, Lutein, Zeaxanthin

AS15 Zeaxanthin, Lycopene, Lutein

AS16 Lycopene, Lutein, Zeaxanthin

AS17 Lycopene, Astaxanthin




�

The HPLC Chromatogram of algal sample AS10 and thei

visible spectrum are shown in

Figure 3.9: HPLC Chromatogram of an extract of algal sample AS1

x 4.6 mm, 5�m column, mobile phase gradient of CH

DCM, detection wavelength 450 nm. Peak no.1 at rete

zeaxanthin and peak no.2 at retention time 14.31 mi

The identification and estimation of

as detailed in the procedure of section 2.7 and 2.8 in C

The peak no.1 (retention time10.12 min) was identif

retention time and visible spectrum

that of the standard zeaxanthin.

The peak 2 (retention time 14.23 min) was identifie


of the authentic sample of lycopene.

The HPLC Chromatogram of algal sample AS10 and their corresponding UV

are shown in Figure 3.9.

��

HPLC Chromatogram of an extract of algal sample AS10 : LC

m column, mobile phase gradient of CH3CN-H2O and CH

DCM, detection wavelength 450 nm. Peak no.1 at retention time 10.12 min is of

zeaxanthin and peak no.2 at retention time 14.31 min is that of lycopene.

The identification and estimation of carotenoids in algal extracts were carried out

dure of section 2.7 and 2.8 in Chapter 2.

The peak no.1 (retention time10.12 min) was identified as zeaxanthin because its

tention time and visible spectrum ( �max at 421, 454, 474 nm) corresponds to

that of the standard zeaxanthin.

The peak 2 (retention time 14.23 min) was identified as lycopene because its

tention time and visible spectrum (�max at 434, 474, 502 nm) resembles to that


�

59

corresponding UV

0 : LC-8 , 25

O and CH3CN-

ntion time 10.12 min is of

carotenoids in algal extracts were carried out

ied as zeaxanthin because its

nm) corresponds to

d as lycopene because its

at 434, 474, 502 nm) resembles to that

�

Table 3.8: HPLC separated carotenoids identified in the algal

their retention times and visible spectrum

Peak number Retention

time in min

1

2

10.12

14.23

Zeaxanthin and lycopene contents were estimated fro

with the corresponding carotenoids. The experiment

The mean and the standared deviation of each carote

The HPLC Chromatogram of algal sample AS12 and their

spectrum are shown in Figure 3.10.

Figure 3.10: HPLC Chromatogram of a

x 4.6mm, 5�m column, mobile phase gradient of CH

DCM, detection wavelength 450 nm. Peak no.1 is of lutein

retention time 14.51 min is that of lycopene.

HPLC separated carotenoids identified in the algal sample AS10 with

ention times and visible spectrum

Retention

time in min

Visible spectrum

(�max) in nm

Identification

10.12

14.23

421, 454, 474

434, 474, 502

Zeaxanthin

Lycopene

Zeaxanthin and lycopene contents were estimated from the peak area associated

with the corresponding carotenoids. The experiment was repeated three times.

The mean and the standared deviation of each carotenoid was worked out.

Chromatogram of algal sample AS12 and their corresponding UV

Figure 3.10.

HPLC Chromatogram of an extract of algal sample AS12: LC

m column, mobile phase gradient of CH3CN-H2O and CH

detection wavelength 450 nm. Peak no.1 is of lutein and peak no.2 at

retention time 14.51 min is that of lycopene.

�

60

sample AS10 with

Identification

Zeaxanthin

Lycopene

m the peak area associated

was repeated three times.

corresponding UV

LC-8,25

O and CH3CN-

and peak no.2 at

�

The peak no.1 (retention time 6.44 min) was identif


of the authentic sample of lutein.




Table 3.9: HPLC separated carotenoids identified in the alga

their retention times and visible spectrum

Peak number Retention

time in min

1

2

6.44

14.51

Lutein and lycopene contents were estimated from th

the corresponding carotenoids. The experiments were

mean and standard deviation of each carotenoid was

The HPLC Chromatogram of the algal sample AS14 is s




The peak no.1 (retention time 6.44 min) was identified as lutein because its

tention time and visible spectrum ( �max at 421, 444, 474 nm) resembles to that

of the authentic sample of lutein.


tention time and visible spectrum (�max at 434, 474, 502 nm) resembles to that


: HPLC separated carotenoids identified in the algal sample AS12 with

ention times and visible spectrum

Retention

time in min

Visible spectrum

( �max) in nm

Identification

6.44

14.51

421, 444, 474

434, 474, 502

Lutein

Lycopene

Lutein and lycopene contents were estimated from the peak area associated with

the corresponding carotenoids. The experiments were repeated three times. The

mean and standard deviation of each carotenoid was worked out.

The HPLC Chromatogram of the algal sample AS14 is shown in Figure 3.11

HPLC Chromatogram of an extract of algal sample AS14


tention time and visible spectrum ( �max at 421, 444, 474 nm).

�

61

ied as lutein because its

resembles to that


at 434, 474, 502 nm) resembles to that

l sample AS12 with

Identification

Lycopene

e peak area associated with

repeated three times. The

Figure 3.11


�

The peak no.2 (retention time10.12 min) was identif


that of the standard zeaxanthin

The peak 3 (retention time 14.51 min)


the authentic sample of lycopene

The HPLC Chromatogram of algal sample AS16 and the

spectrum are shown in Figure 3.


The peak no.2 (retention time10.12 min) was identified as zeaxanthin because its




tention time and visible spectrum (�max at 434, 474, 502 nm) resembles that of



Figure 3.12.

��


�

62

ied as zeaxanthin because its

at 421, 454, 474 nm) corresponds to

was identified as lycopene because its

at 434, 474, 502 nm) resembles that of

ir corresponding UV


�



The peak no.2 (retention time11.66 min) was identified as zeaxanthin because






The HPLC Chromatogram of algal sample AS11 an

spectrum are shown in Figure 3.13

Figure 3.13: HPLC Chromatogram of saponified extract of

Column: LC-8, 25 x 4.6mm, 5

H2O and CH3CN-DCM, detection wavelength 450 nm.


tention time and visible spectrum ( �max at 421, 445, 470 nm).

time11.66 min) was identified as zeaxanthin because




me and visible spectrum (�max at 434, 474, 501 nm) resembles that of



Figure 3.13

HPLC Chromatogram of saponified extract of algal sample AS11:

8, 25 x 4.6mm, 5�m column, mobile phase gradient of CH

DCM, detection wavelength 450 nm.

�

63


time11.66 min) was identified as zeaxanthin because its

at 421, 461, 489 nm) corresponds to


at 434, 474, 501 nm) resembles that of

d their corresponding UV

algal sample AS11:

m column, mobile phase gradient of CH3CN -

�

Table 3.10 : HPLC seperated carotenoids identified in the alga

Peak

number

Retention time

in min

1

2

3

4

6.44

7.54

8.18

14.51

The HPLC Chromatogram of algal sample AS5 and the

spectrum are shown in Figure 3.14.



retention time and visible spectraum

: HPLC seperated carotenoids identified in the algal sample AS 11

Retention time

in min

Visible spectrum(�max )

in nm

Identification

14.51

421, 444, 474

485

472

434, 474, 502

Lutein

Astaxanthin

Cis -Astaxanthin

Lycopene


Figure 3.14.



ention time and visible spectraum( �max at 421, 449, 479 nm).

�

64

AS 11

Identification

Lutein

Astaxanthin

Astaxanthin

Lycopene

corresponding UV



�

65 �

The peak no. 2 (retention time 13.64 min) was identified as lycopene because its

retention time and visible spectrum (�max at 434, 474, 501 nm) resembles that of


The amount of different carotenoids estimated from the peak area in different

algal samples are shown in Table 3.11

Table 3.11: Estimated carotenoid percentage in different algal samples

Sample

ID

Total carotenoid

mg/g

% of carotenoids

Lycopene Lutein Astaxanthin Zeaxanthin

AS1 6.33±0.31 85 10 -- --

AS2 9.26±0.22 35 -- 60 --

AS3 4.45±0.21 82 13 -- --

AS4 10.24±0.23 67 ---- 18 --

AS5 4.60±0.15 83 14 -- --

AS6 8.97±0.20 86 13

AS7 11.57±0.34 84 14 -- --

AS8 8.60±0.35 86 13 -- --

AS9 6.64±0.42 84 -- -- 12

AS10 23.85±0.36 86 -- -- 11

AS11 9.82±0.26 10 8 65 --

AS12 17.79±0.50 83 12 -- --

AS13 18.2±0.31 12 11 62 --

AS14 9.68±0.41 76 7 -- 8

AS15 6.39±0.50 15 6 -- 74

AS16 23.12±0.41 73 8 -- 12

AS17 11.6±0.25 58 --- 39 --

AS18 9.23±0.51 79 -- -- 11

AS19 11.42±0.24 18 7 58 ---

AS20 11.70±0.35 67 15 -- --

Results are shown as mean ± SD of three experiments

�

66 �

3.8 Separation of geometrical isomers of lycopene by YMC carotenoid

column

The carotenoids extracted from Euglena sp1 was purified by column

chromatography using alumina as adsorbent. The first fraction was collected by

eluting with petroleum ether as the mobile phase. This fraction showed visible

spectra similar to that of standard lycopene (434, 473, 504).

The cis- trans isomer of column purified lycopene fraction was then seperated by

HPLC using YMC carotenoid column ( 250 x 4.6 mm, 5 �m ) with an isocratic

solvent system using the mobile phase as BuOH: CH3CN: DCM= 3:7:1 at the

flow rate of 2 mL min-1

. SPD- M10 AVP Shimadzu Photo Diode Array detector

was set at 470 nm. All trans-lycopene was identified by comparing the retention

time and absorption spectrum with standard lycopene. The cis lycopene was

tentatively identified based on the following rules (Lee et al. 2001).

The mono cis- isomers of lycopene results in a hypsochromic shift of about 4 nm

when compared to the parent all- trans forms.

The central cis isomers of lycopene such as 9'-cis, 5'- cis, 13' cis and 15'- cis have

a strong peak in the UV region at about 340 nm.

The cis isomers of lycopene have smaller extinction coefficients and reduced fine

structure.

The mono- cis or di cis isomers of lycopene could be tentavely identified by the

Q ratio. The Q ratio is defined as the height ratio of the cis peak to the main

absorption peak.

The di-cis – isomers of lycopene may be shifted to shorter wavelenth than its

mono cis form.

The estimation of cis-isomers of lycopene were carried out considering the cis

and trans isomers show equivalent area.

The column purified lycopene content was further seperated into cis and trans

components in a YMC carotenoid column. One chromatogram is shown in

�

67 �

Figure 3.15 while Table 3.12 shows all the separated components along with

their physical parameters.

Figure 3.15 HPLC Chromatogram of lycopene isomers using an YMC column

in an isocratic solvent system using the mobile phase as BuOH: CH3CN:

DCM=3:7:1, detection wave length 470 nm.

Peak no. 1 with retention time 4.654 min showed absorption spectrum at 466, 490

nm is di-cis isomer of lycopene.

Peak no.2 (with retention time 6.182 min and absoption spectrum 382, 446, 469,

490 nm and Q value 0.68) was identified as 15'-cis lycopene.

Peak no.3 (with retention time 6.805 min, absorption spectrum 383, 446, 469,

500 nm and Q – value 0.57 was identified as 13' cis-lycopene.

Peak no.4 (with retention time 8.326 min and absorption spectrum 446, 466, 490

nm) was identified as di-cis-lycopene.

Peak no.5 ( with retention time 9.331 min and absorption spectrum 446, 476, 506

nm) was identified as all trans-lycopene.

Peak no.6 ( with retention time 10.875 min and absorption spectrum 386, 446,

474, 506 nm) was identified as 5'- cis- lycopene.

Peak no. 7 ( with retention time 12.24 min and absorption spectrum 387, 446,

470, 496 nm and Q value 0.28 ) was identified as 9'- cis lycopene.

Peak no. 8 (with retention time 14.49 min and absorption spectrum 329, 440, 465,

490 nm and Q value 0.13 ) was identified as di- cis- lycopene.

�

68 �

The physical properties of all the seperated isomers of lycopene are tabulated in

Table 3.12.

Table 3.12: HPLC separated isomers of lycopene with their retention times,

visible spectra and Q values.

Peak

no.

Isomer of

lycopene

Retention

time (min)

�max

in nm

Q-value

1

2

3

4

5

6

7

8

di-cis - lycopene

15'-cis-lycopene

13'-cis- lycopene

di-cis- lycopene

all-trans- lycopene

5'-cis -lycopene

9'-cis-lycopene

di-cis- lycopene

4.671

6.185

6.804

8.326

9.331

10.875

12.246

14.491

466, 490

382, 446, 469, 490

383, 446, 469, 500

446, 466, 490

446, 476, 506

386, 446, 474, 506

387, 446, 470, 496

329, 440, 465, 490

------

0.68

0.57

------

------

0.40

0.28

0.13

The estimation of different isomers were carried out from the area of the

corresponding peak (considering the cis isomers show similar peak area that of

all-trans isomer of lycopene). The amount of geometrical isomers of lycopene

estimated in Euglena sp1 collected from different water bodies are listed in Table

3.13.

�

69 �

di-

cis

-

lyco

pene

(mg

/g)

0.1

06±

0.0

014

0.2

64±

0.0

015

0.3

12±

0.0

013

0.6

22±

0.0

012

0.4

12±

0.0

016

0.1

54±

0.0

015

9'-

cis

-

lyco

pen

e

(mg

/g)

0.6

13±

0.0

017

0.5

01±

0.0

01

1.2

15±

0.0

014

3.9

54±

0.0

015

2.5

50±

0.0

014

0.5

81±

0.0

016

5'-

cis

-

lyco

pen

e

(mg

/g)

0.0

25

±0.0

01

8

0.1

43

±0.0

01

5

0.1

16

±0.0

01

6

0.3

61

±0.0

01

5

0.2

11

±0.0

01

6

0.2

77

±0.0

01

4

All

tr

an

s-

lyco

pene

(mg

/g)

3.8

21±

0.0

021

4.1

93±

0.0

016

6.5

14±

0.0

022

12.0

13±

0.0

02

1

11.5

01±

0.0

03

1

5.0

21±

0.0

021

di-

cis

-

lyco

pen

e

(mg

/g)

0.3

02±

0.0

014

0.6

82±

0.0

012

0.4

52±

0.0

013

1.2

81±

0.0

013

0.6

21±

0.0

012

0.2

24

1±

0.0

014

13

'-cis

-

lyco

pen

e

(mg

/g)

0.4

61

±0.0

01

3

0.8

12

±0.0

01

2

0.5

62

±0.0

01

4

1.4

63

±0.0

01

2

1.2

33

±0.0

01

5

0.4

71

±0.0

01

5

15

'-cis

-

lyco

pen

e

(mg

/g)

0.0

20±

0.0

011

0.1

82±

0.0

015

0.2

06±

0.0

013

0.5

33±

0.0

012

0.3

44±

0.0

014

0.2

02±

0.0

012

di-

cis

-

lyco

pene

(mg

/g)

0.0

13±

0.0

012

0.0

83±

0.0

012

0.0

41±

0.0

013

0.2

52±

0.0

012

0.1

92

± 0

.0015

0.1

43±

0.0

014

Alg

al

sou

rce

AS

1

AS

4

AS

7

AS

10

AS

16

AS

18

�

NMR data: The NMR spectrum

sample AS10 is shown in Figure 3.16

was seperated by column chromatography and then by

fraction was collected in DCM which was evaporated

CDCl3 and then the NMR spectrum

Figure 3.16: HNMR spectrum

500.13 MHz

The proton NMR spectrum

to 2 ppm due to the methyl group in lycopene. Thes

13' –CH3, 1,1

' -CH3, 5, 5

'- CH

betwween 4.200 to 4.232 ppm are due to

The NMR spectrum of pure lycopene extracted from the algal

Figure 3.16. The saponified extract of the algal sample

was seperated by column chromatography and then by TLC. The TLC seperated

fraction was collected in DCM which was evaporated to dryness and dissolved in

and then the NMR spectrum was recorded.

HNMR spectrum of TLC purified lycopene extract recorded in

The proton NMR spectrum of pure lycopene showed strong singlet betwwen 0.8

to 2 ppm due to the methyl group in lycopene. These are due to the 9,9'-CH

CH3. Peaks at 2.17 ppm was due to 3, 3' CH2 and peaks

betwween 4.200 to 4.232 ppm are due to olefinic proton.

�

70

of pure lycopene extracted from the algal

The saponified extract of the algal sample

TLC. The TLC seperated

ss and dissolved in

of TLC purified lycopene extract recorded in

of pure lycopene showed strong singlet betwwen 0.8

CH3, 13,

and peaks

�

C13

NMR spectrum of pure lycopene extracted from algal sample AS10 i

in Figure 3.17.

Figure 3.17: C13

NMR spectrum

AS10

In the C13

NMR spectrum

ppm is due to the olefinic carbon, peak between 76.

and the peak between 23 to 38.7 ppm are due to CH

IR spectrum of TLC purified lycopene extracted from the algal s

shown in Figure 3.18

Figure 3.18: The IR spectrum

AS10 recorded using KBr pellet.

of pure lycopene extracted from algal sample AS10 is shown

NMR spectrum of pure lycopene extract from algal sample

NMR spectrum of lycopene the observed peak between 128.8

ppm is due to the olefinic carbon, peak between 76.6 -77.4 are due to CH2

and the peak between 23 to 38.7 ppm are due to CH3 carbon.

of TLC purified lycopene extracted from the algal sample AS10 is

The IR spectrum of pure lycopene extracted from algal sample

AS10 recorded using KBr pellet.

�

71

of pure lycopene extracted from algal sample AS10 is shown

of pure lycopene extract from algal sample

of lycopene the observed peak between 128.8-132.42

2 carbon

ample AS10 is

of pure lycopene extracted from algal sample

�

In the IR spectrum the peak at 2958.8 cm

the peak at 2924.09 (s) cm

group. The peak at 1095.57 cm

NMR data of astaxanthin

from the algal sample AS1

the algal sample was seperated by column chromatogr

TLC seperated fraction was collected in DCM which w

and dissolved in CDCl3 and then the NMR spectra was recorded

Figure 3.19: The HNMR spectrum

Figure 3.20: IR spectrum of pure a

IR data of astaxanthin in KBr pellet:

is due to C=O cm-1

stretching, the peaks at 2927.94

to C-H stretching, the peaks at 3441.01 cm

In the IR spectrum the peak at 2958.8 cm-1

is due to the olefinic C-H stretching,

the peak at 2924.09 (s) cm-1

and 2854.65 cm-1

are due to C-H stretching of CH

group. The peak at 1095.57 cm-1

is due to C-C stretching.

astaxanthin: The NMR spectrum of pure astaxanthin extracted

from the algal sample AS11 is shown in Figure 3.19. The saponified extract of

the algal sample was seperated by column chromatography and then by TLC. The

TLC seperated fraction was collected in DCM which was evaporated to dryness

and then the NMR spectra was recorded.

HNMR spectrum of astaxanthin recorded in 500.13MHz

of pure astaxanthin extracted from algal sample AS11

staxanthin in KBr pellet: In IR spectrum the peaks at 1716.65 cm

stretching, the peaks at 2927.94 cm-1

and 2858.51 cm-1

H stretching, the peaks at 3441.01 cm-1

is due to the O-H stretching, 1639.49

�

72

H stretching,

H stretching of CH3

extracted

he saponified extract of

aphy and then by TLC. The

as evaporated to dryness

500.13MHz

staxanthin extracted from algal sample AS11

In IR spectrum the peaks at 1716.65 cm-1

are due

H stretching, 1639.49

�

73 �

cm-1

is due to C=C stretching, 1446.61 cm-1

is due to C-H deforming and the peak

at 1180.44 cm-1

is due to C-C stretching of astaxanthin

Identification of carotenoids by LCMS: The LCMS spectrum of some algal

samples were recorded using Agilent 6410 Triple Quad LC MS-MS (with Agilent

1260 Infinity Series HPLC system). Detector: MS (As in LCMS system the MS

itself is the detector), Column: Zorbax Reverse Phase C-18 Column (4.6 x 250

mm). The solvent system used for LCMS analysis of algal samples was

Acetonitrile: Water with mode: ESI.

LCMS spectrum of TLC purified of pure astaxanthin extracted from the algal

sample AS11 is shown in Figure 3.21.

Figure 3.21: LCMS spectrum of astaxanthin

�

74 �

In the LC-MS mass chromatogram the fraction eluted at 15.654 min having m/z

=579.2 was identified as 13-cis astaxanthin and the fraction eluted at 21.672 min

having m/z=579.3 was identified as all trans astanxanthin. The m/z=579.3 is due

to the fragment ion of [M+H-H2O] +

.

LCMS spectrum of TLC purified lycopene extracted from the algal sample AS10

is shown in Figure 3.22

Figure 3.22: LCMS spectrum of TLC purified lycopene

The fraction eluted at 21.399 min having m/z =536.4 was identified as lycopene

due to the molecular ion peak [M+H] +

.

3.9 Separation of algal extract by HPTLC

The saponified extract of some algal samples were separated and identified by

HPTLC. Aluminum supported TLC plates (silica gel-G 60 F254) of size 20 × 20

cm were cut into 4 equal plates of size 10 × 10 cm. The plates were prewashed

with methanol and dried in oven for 20 minutes at 85ºC. The sample and standard

compounds were applied to the layers by means of a Linomat-5 applicator as

bands of 6 mm wide at the height of 10 mm from the base. The plate was

developed in a twin trough chamber of size 20 × 10 cm using 12 mL respective

mobile phase for different markers. Before the plate development, the mobile

phase was allowed to saturate in the tank for 15 minutes. After the development,

�

75 �

the plate was dried by hair dryer for complete evaporation of mobile phase and

scanned at multiple wavelengths using CAMAG TLC Scanner equipped with

winCATS software. The plate was again scanned at multiple wavelengths (200

nm-700 nm) using CAMAG TLC Scanner at slit dimension 6.00 x 0.45 mm

micro with a scanning speed 20 mm/s in absorption-reflection detection mode.

Figure 3.23: HPTLC chromatogram of Algal sample AS4 and AS17

�

�

76

�

�

�

�

77

�

78 �

Figure 3.24: HPTLC spectrum of algal extract

In the HPTLC chromatogramme, the track 1,2,3,4 and 5 are standard astaxanthin

at different concentrations. The Rf value of standard astaxanthin was measured at

0.37. Track 8 is the standard lycopene showing Rf value at 0.79. the solvent used

here was methanol. Track 6 is the algal sample AS4. It shows two major

carotenoids lycopene and astaxanthin. The lycopene content was estimated to be

67% and astaxanthin was 18% and the rest is unidentified carotenoid. The track 7

is that of the algal sample AS17, with two major fractions, having 58.57%

lycopene and 39.63% of astaxanthin.

3.10 Influence of Seasonal variation on carotenoid production by algae in

natural condition: The production of carotenoids in algae depends on intensity

of light on which a particular alga grows (Edwards et al. 2006). As the intensity

of light in natural condition varies seasonally, so the algal growth and the

carotenoid production in different seasons in a year may vary accordingly. For

this reason, in the present experiment six algal samples (AS7, AS10, AS11, AS

12, AS13, and AS16) were randomly selected for analysis of carotenoid content

in different seasons throughout the year. The algal samples from the selected

ponds were collected in the months of February, April, June, August and

December. The collected algal samples were washed and freeze dried. From the

dried samples carotenoids were extracted and then estimated using

Spectrophotometer. The amount of total carotenoids estimated in different

seasons are shown in Table 3.14

�

79 �

Table 3.14: Seasonal variation of carotenoid in algae in natural condition

Source Month Total carotenoids

(mg/g)

Major carotenoid

AS7

February 8.12±0.31

Lycopene

April 8.50±0.23

June 10.34±0.53

August 11.57±0.34

December 9.72±0.42

AS 10

February 16.3 ±0.24

Lycopene

April 17.4±0.23

June 19.3±0.36

August 23.85±0.36

December 15.4±0.28

AS11

February 4.50±0.24

Astaxanthin

April 4.60±0.34

June 6.8 ±0.52

August 9.82±0.26

December 6.60 ±0.38

AS12

February 12.42±0.63

Lycopene

April 10.11±0.23

June 14.22±0.25

August 17.79±0.50

December 13.86±0.44

AS13


Astaxanthin

April 14.43±0.36

June 15.40±0.43

August 18.2±0.31

December 15.3±±0.55

AS16


Lycopene

April 17.83±0.62

June 18.56±0.54

August 23.12±0.41

December 18.96±0.64


�

80 �

It was confirmed from the study that algae collected from the fishery ponds

contains a good amount of carotenoids. A maximum of 23.8 mg/g carotenoids of

dry biomass of algae was estimated, where the major carotenoid was found to be

lycopene. Seasonal variation of carotenoid production in six ponds were analyzed

during the months of February, April, June, August and December where it was

found that carotenoid production was highest in the month of August. This may

be due the highest light intensity during this period and favorable temperature

range available at this time. Seasonal variation of fresh water algae was noticed

during the study. The least production of carotenoids was found in the month of

February. During the month of April, least algal bloom was found yet the total

carotenoid was found to be little more than that during the month of February.

This may be due to the fact that during the month of April the pH of pond water

was found to be more, as in this season lime is used in the fishery ponds to

protect fishes from different diseases. At the higher pH algal growth decreases

and so the algal bloom decreases (Ritcher et al. 2003). Also in this period salinity

of pond water becomes less due to heavy rains, so the algal forms showed

considerable variations in their stress metabolites concentrations as well as

enzymatic activity with respect to the changes in salinity pattern in the natural

condition of the algal growth. These results are in agreement with Chakraborty et

al. 2010, who reported the decrease in salinity in monsoon during the month of

August.

3.11 Influence of the pond water pH and carotenoid content in algae: The

algal growth is depended on the pH of the media. The pond water samples were

collected from where the algae samples were taken and the pH of the water

samples was measured with the help of a digital pH meter. The total carotenoids

were also estimated in the algal samples grown in different pond water, so that

any influence of water pH on algal carotenoid synthesis could be observed. The

estimated total carotenoids and the corresponding pH of the different algal

samples are shown the Table 3.15.

�

81 �

Table 3.15: Effect of carotenoid content with different pH of the pond water

Sample ID pH of the pond water Total carotenoid ( mg/g)

AS1 6.40 6.33±0.31

AS2 6.82 9.26±0.22

AS3 7.81 4.45±0.21

AS4 6.94 10.24±0.23

AS5 6.41 4.60±0.15

AS6 9.63 8.97±0.20

AS7 9.64 11.57±0.34

AS8 9.80 8.60±0.35

AS9 6.52 6.64±0.42

AS10 6.80 23.85±0.36

AS11 6.92 9.82±0.26

AS12 7.26 17.79±0.50

AS13 7.22 18.2±0.31

AS14 6.93 9.68±0.41

AS15 7.99 6.39±0.50

AS16 7.40 23.12±0.41

AS17 7.22 11.6±0.25

AS18 10.65 9.23±0.51

AS19 6.81 11.42±0.24

AS20 6.85 11.7±0.35


�

82 �

3.12 DISCUSSION

The red algal scum collected from different natural sources comprising three

districts (Kamrup, Morigaon and Darrang) of Assam, was found to be very rich

source of carotenoids. For collection of algae twenty different ponds (AS1-AS20)

were selected randomly from different locations. The algae collected from these

ponds were found to be Euglena genera having two species, which are named as

Euglena sp1 and Euglena sp2. Out of the twenty selected ponds, Euglena sp1 was

found in fourteen ponds, whereas six ponds (AS2, AS4, AS11, AS13, AS17, and

AS19) are found to be rich in Euglena sp2. Euglena sp1 was found to be rich in

lycopene whereas astaxanthin was found in Euglena sp2. The total carotenoid

estimated in each algal sample was found to be different and depended upon the

collection source (Table 3.1). The total carotenoids content in Euglena sp1 in

different sources are found to be in the range of 6.33-23.85 mg/g of dry algal

biomass. A maximum of 23.85 mg of carotenoids per gm of dry weight of algae

was estimated in the algal sample AS10, of which lycopene content was found to

be as high as 20.5 mg per gm of dry algae. This amount is far more than the

lycopene content of tomato. The variation of total carotenoids in different

environmental conditions of algae may be due to the varied nutrient level, pH of

the water media and difference in exposure to sun light. HPLC analysis showed

that algal sample AS10 contains lycopene as the major carotenoid. A small

amount of zeaxanthin was also estimated in this alga found in the natural

conditions. From HPLC data it was estimated that the algal sample AS10

contains 86% of lycopene and 11% of zeaxanthin. The high lycopene content

may be due to the absence or inhibition of cyclase enzymes, which converts

lycopene into other cyclic carotenoids (Zhong et al. 2011). There is always a

possibility that the pond water quality may prevent these lycopene cyclase

enzymes from its activity. The carotenoid biosynthetic pathways are reported by

many authors. Umeno et al. 2005 reported the biosynthesis of lycopene from the

bio precursor isopentyl pyrophosphate (IPP) and subsequent synthesis of

astaxanthin and other xanthophylls by different enzymes present in yeast, bacteria

and algae (Britton et al.1995, Fraser et al.1997). The pH of water in the algal

source AS10 was 6.8 in which the lycopene cyclase enzyme may be inactive for

which the lycopene was found to be as the major carotenoid. In the algae Euglena

�

83 �

sp2 the total carotenoids estimated in different sources is found to be in the range

of 9.26-18.2 mg/g of dry algal biomass. Euglena sp2 collected from the pond

AS13 was found to contain 18.2 mg/g of total carotenoids. The pH measurement

of this pond water was found to be 7.22. The percentage of astaxanthin in this

algal sample was found to be 62% along with 12% lycopene and 11% lutein.

Heavy algal bloom of Euglena sp2 was found in the case of algal sample AS11,

but the total carotenoids were found to be less than that of algal sample AS13.

The total carotenoid in the algal sample AS11 was estimated to be 9.82 mg/g of

dry weight of algae of which astaxanthin content was 65%. The pH of this pond

water was found to be 6.92. The higher percentage of astaxanthin in this sample

may be due to the presence of suitable active enzymes, which facilitates the

biosynthesis of astaxanthin.

The pH measurement of the pond water is also carried out, which shows some

correlation of growth of algae and the pH of the pond water media. Euglena sp1

bloom was found where the pond water pH was in the range of 6.40-10.65.

Although this algae can tolerate wide range of pH yet the heavy algal bloom was

found in the acidic media in the pond AS10 where the water pH was 6.80. In case

of Euglena sp1 the correlation coefficient of pH and total carotenoid content was

found to be -0.1108. This shows that total carotenoids accumulation in Euglena

sp1 is not directly associated with pH of the media but also depends on other

environmental conditions. The correlation coefficient of pH and carotenoids

content of Euglena sp2 was found to be 0.938, which indicates that pH of algal

growth media has positive impact on carotenoid accumulation in this algal

species. The algal bloom of the Euglena sp2 was found where the pH of the pond

water was in the range of 6.82-7.22. Heavy algal bloom of this alga was found in

the pond AS11 where the water pH was 6.92. However the total carotenoid in this

alga was found to be higher (18.2 mg/g) in pond AS13 where the water pH was

7.22. Thus the algal growth and carotenoid production are not inter-related i.e.

more algal biomass not necessarily the more carotenoid accumulation.

Carotenoid estimation in these algae shows that the maximum carotenoids were

produced by Euglena sp1 when the pH of the pond water is around pH 6.8. This

result was supported by Rahman et al. (2007) and Olaveson et al. (2000). They

�

84 �

found that Euglena mutabilis and Euglena gracilis were acid tolerant, growing

optimally at pH 2.5 to 7.0. The pH tolerances of different algae are different.

Baumer et al. (2001) reported the cultivation of Euglena gracilis at pH 6.5-8.0.

Dayananda et al. (2010) reported the biomass production and carotenoid

production in microalgae at different pH where the maximum yield was recorded

at pH 7.5. In the present study, it was found that a pH of around 6.8 is most

conducive to the heavy bloom of Euglenophytes. Furthermore, Xavier et al.

(1991) reported that Euglena sanguinea bloom developed when the pH value was

around 6.9.

Seasonal variations of carotenoid content in the algal samples were studied. In

our study six algal samples (ponds AS7, AS10, AS11, AS12, AS13, and AS16)

were randomly selected for analysis of carotenoid content in different seasons

throughout the year. Algae were collected from these ponds in the months of

February, April, June August and December and the total carotenoids were

estimated using UV Visible spectrophotometer (Table 3.4). The results showed

that the maximum carotenoid production was found during the month of August

and the least carotenoids production was found during the month of February. As

the day light intensity in the North Eastern part of India is generally more during

the month of August, so we may conclude that high light intensity facilitates the

high carotenoid synthesis during the month of August and in the month of

February the light intensity is less so the carotenoid production is less during this

period. These results are in agreement with the report of Edwards et al. 2006. As

the algal biomass and carotenoid production of a particular alga was found to be

different in different algal source, so the algae from the pond water AS10 and

AS11 were cultured under different nutrient stress condition and their

corresponding carotenoid profiles were studied in Chapter 4.

Studies in human animal models support that cis- lycopene is better absorbed

than all-trans- isomers. Fresh tomato and their products contain about 90%

lycopene in all trans- form (Clinton et al. 1996, Gartner et al.1997). But the algae

we have analyzed contain about 58.57% of all trans and rest 41.24% is in cis-

form. Further in vitro studies indicate that cis-isomers are preferentially absorbed

in mixed micelles in the intestine and are more bioavailable (Boileau et al. 1999).

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Though it has been reported that a minor amount of all-trans- lycopene might be

converted to cis- form in the stomach and other tissues, a source with more than

40% cis- form would be certainly advantage with respect to bioavailability.

Therefore lycopene from Euglena may be better choice in this aspect when

compared to its common source as tomato.

characterization of carotenoids in algae collected...

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