CHLOROPHYLL

PAPER – 1:
 Half a gram of freeze-dried algae was thoroughly mixed with 5 mL of methanol and
incubated at 45 °C for 24 h.
 The resultant mixture was centrifuged at 4500 rpm for 10 min.
 The absorbance of the supernatant was measured at 470, 652.4, and 665.2 nm using a
spectrophotometer (Thermo Scientific)

Chlorophyll a = 16.72 (A665.2) − 9.16 (A652.4) …………………………. (1)

Chlorophyll b = 34.09 (A652.4) − 15.28 (A665.2) (3) ……………………. (2)

Total Chlorophyll = Chlorophyll a + Chlorophyll b (4) …………………… (3)

PAPER - 2:

 To determine the amount of pigment present in microalgae.

 2 mL microalgal culture was centrifuged at 6000 rpm for 5 min.
 The methanol was used to dissolve the cell pellet, and then it was kept overnight at 4 °C.
 Further, the pigment composition (chl-a, chl-b, and carotenoids) was determined using
the following formulas after measuring the absorbance of the supernatant at 665.2, 652.4,
and 470 nm, respectively.

Chlorophyll a: Chl-a (µg/mL) = 16.72 * A665.2 - 9.16 * A652.4

Chlorophyll b: Chl-b (µg/mL) = 34.09 * A652.4 - 15.28 * A665.2

Carotenoid (µg/mL) = (1000 * A470 - 1.63 * Chl-a - 104.9 * Chl-b) / 221

PAPER – 3:

 For the total carotenoids and PBPs, a sequential extraction was performed with ethanol
 (96%, v/v) and water, respectively, on days 0, 1, 4, 6, 8, 11, 15, 18, 20, and 22.
 These extracts were also used to ascertain the antioxidant capacity.
 To analyze the total carotenoid content, 3 mL aliquots of different culture conditions were
harvested and centrifuged (2744 * g for 10 min);
 The supernatant was discarded, and 3 mL of ethanol was added to the pellet with glass
beads.
 Each sample was extracted in a Precellys homogenizer (Bertin Technologies, Montigny-
le-Bretonneux, France), using a 6 min cycle at 8000 rpm (30 s homogenization with 40 s
stopping intervals).
 Ethanolic extracts were centrifuged at 2744 * g for 10 min, kept under dimmed light, and
stored at -4 ° C until pigment content and antioxidant capacity determination.
 The total carotenoid content was determined spectrophotometrically (Shimadzu UV-
1800, Kyoto, Japan) according to Lichtenthaler and Buschmann (2001) [22].
 The absorption was read at λ = 470, λ = 664, and λ = 648 nm, and the content was
calculated as follows:

Chl a (µg. mL-1) = (13.36 A664) - (5.19 A648) ………………………….. (1)

Total carotenoids (µg. mL-1) = (1000 A470) - (2.13 Chl a) / 209 ………………. (2)

 For the extraction of total PBPs, the remaining pellet of ethanolic extraction was added
with 3 mL of water, and then samples were subjected to vortexing for 20 s.
 The extract was centrifuged at 2744* g for 8 min and then kept under dark conditions
before the analysis for pigment content and antioxidant capacity.
 The determination of the PBP content, namely phycocyanin (PC), allophycocyanin
(APC), and phycoerythrin (PE), was via the spectrophotometric measurement of
absorbance
  at λ = 562, 615, and 652 nm, followed by the application of Bennett and Bogorads
equations, see Equations (3)–(5):

PC (mg. mL-1) = (A615) – 0.474 A652) / 5.34 …………….. (3)

APC (mg. mL-1) = [ (A652) – (0.208 A615)] / 5.09 ……….. (4)

PE (mg. mL-1) = [(A562) – 2.41(PC) – 0.849 (APC)] / 9.62 …… (5)

The PBP productivities were expressed in mg. L-1 .d-1

PAPER -4:

 Chlorophyll a was spectrophotometrically estimated in methanol (80%) and calculated

according to Metzner et al. (1965).
 Using the following formula, the growth rate (μ) measured as chl. a was calculated:

μ (h−1) = (LnA2−LnA1) / (t2−t1) ………………… (1)

where A2 and A1 indicate the concentrations of chl. a at t2 (day 10) and t1 (day 0), respectively.
The generation time (G) can be calculated as follows: G = ln2/μ.

PAPER – 5:

 Chlorophyll estimation was carried out by harvesting 5 mL culture from each

experimental flask.
 Twice distilled water wash was followed by the resuspension of the cell pellet in 5 mL
absolute ethanol (95%).
 Incubation of 5 min at 100 ◦C in a water bath was given to the samples after addition of
ethanol. Supernatants’ absorbance was taken at 645 and 663 nm for computing the
chlorophyll content.
PAPER – 6:
 The pigment profiles were identified according to the method described by Ji et al.
 Briefly, the extract was analyzed at three optical densities (OD) of 665 nm, 649 nm, and
470 nm using a spectrophotometer (HACH DR 3900, Loveland, Co.).
 The chlorophyll concentration was calculated using the following equation:

Chla = 13.7 × OD665 - 5.76 × OD649.

The chlorophyll b (Chlb) concentration was calculated using the following equation:

Chlb = 24.96 × OD649 - 7.32 × OD665.

The carotenoid concentration was calculated using the following equation:

Carotenoids = (1000 × OD470 - 2.05 × Chla) / 245.

PAPER – 7:
 To estimate the Chlorophyll-a content in the microalgae the colorimetry method has been
widely used.
 The microalgae were centrifuged at 6000 rpm for minimum of 10 min and washed with
deionized water to remove impurities and resuspended in the methanol.
 Later, they treated in the water bath at 60 ◦C for 45 mins for extraction.
 The obtained Chlorophyll-a cooled at room temperature.
 Using the Porras equation, the Chl-a concentration were estimated.

Chl-a = 16.29 (A665.2 – A750) – 8.54 (A652 – A750)

Where, (A652 – A750) and (A665.2 – A750) are the absorbance of the chlorophyll.