Chapter 3: Materials and Methods

 Area of seaweed collection

The seaweed samples were collected during 2015-2017. Different seaweed
species of Ulva lactuca, Ulva reticulata, Gracillaria corticata, Kappaphycus
alvarezii, Sargassum johnstonii and Padina pavonica from belonging to
Chlorophyceae, Rhodophyceae and Pheophyceae families were collected from Okha
coast and Beyt-dwarka. Okha coast and Beyt-dwarka, is situated at 22° 28' 7.3272'' N
& 69° 4' 11.3664'' E 22° 27' 24.7644" N & 69° 5' 41.5968" E in the “Gulf of Kutch”
on the north- western most part of Saurashtra in Gujarat, India (Plate 4). These areas
are one of the most important places of interest for marine algal growth in India. After
collection of seaweeds, its washed immediately with seawater to remove other debris,
other epiphytes and sand particles. Then it was transferred in laboratory and washed
thoroughly with simple water up to 3 to 4 times for removal of extra salts on the
surface. After that, seaweeds were identified by following book of “Study of seaweed
diversity along the island of gulf of Kachchh, Gujarat” (Gujarat Ecology
Commission)240 and help of fisheries department, Junagadh Agriculture University
(JAU), Okha, Gujarat.
 Collection of vegetable seeds
The seeds of Solanum melongena L., Lycopersicon esculentum Mill.,
Capsicum annuum L., Brassica oleracea var. Capitata L. and Allium cepa L. were
collected from Vegetable Scientific research center, Anand Agriculture University,
Anand, Gujarat and Gandhi Agro, Anand during 2015-2017 (Plate 5).
 Preparation of Seaweed Liquid Fertilizer (SLF)
The seaweeds of Ulva lactuca (A1), Ulva reticulata (A2), Gracillaria
corticata (A3), Kappaphycus alvarezii (A4), Sargassum johnstonii (A5) and Padina
pavonica (A6) were dried under sunlight. Then after, the seaweed was grinded and
prepared its powder form. 1kg powder of each seaweed species was mixed with 20 L
of water in the ratio of 1:20. The mixture was boiled for 1 h and cooled. The cooled
mixture was sifted with muslin fabric51. The obtained seaweed liquid extract was
considered as 100% concentration of standard solution and different concentration
was prepared from diluting with water of seaweed liquid fertilizer. In this study, also
prepared the mixture of both green (A1+A2), red (A3+A4) and brown (A5+A6) and
all selected seaweed mixture for seaweed liquid fertilizer (AM).

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Plate 4

Map of Okha Coast and Beyt -Dwarka, Gujarat, India

A- Okha cost B- Beyt- Dwarka coast

A and B- Collection site of seaweed

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Plate 5

Solanum melongena L. Lycopersicon esculentum Mill.

Capsicum annuum L.

Brassica oleracea var. Capitata L. Allium cepa L.

Seeds of selected vegetables

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 Estimation of physico-chemical parameters, macro & micro

nutrients and growth hormone of seaweed liquid fertilizer (SLF)
pH, Electrical Conductivity & Organic Carbon (C) and primary macro
nutrients of Total Nitrogen (N), Total Phosphorus (P) & Total Potassium (K) analysis
of seaweed liquid fertilizer (SLF) were carried out by standard method22, 252. Above
analysis was done in laboratory at V. P. & R. P. T. P. Science College, Vallabh
Vidyanagar. Whereas, secondary macro nutrients of S (sulphur), Ca (calcium) & Mg
(magnesium) analysis was done at Bail & SICART, respectively. Other
micronutrients such as B (boron), Cu (copper), Mo (molybdenum), Zn (zinc) & Mn
(manganese) were analyzed at SICART by ICP-OES (Inductively Coupled Plasma-
Optical Emission Spectrometer, Perkin Elmer, Optima 3300 RL). Auxin and
gibberelline (plant growth promoting hormones) was analyzed at Junagadh
Agriculture University, Junagadh.

 Estimation of Physico-chemical parameters

1) pH (Potentiometric method)
Principle:

The main principle of electronic pH measurement of acidity or alkalinity of a solution

is the determination of activity of hydrogen ion by potentiometric measurement using
a standard sensing electrode. pH was measured by a Digital pH meter (Digital pH,
conductivity & temperature meter, 181) its electrode was washed twice with double
distilled water (DDW) before standardization.

Calibration

pH meter was calibrated by sinking the electrode in standard buffer solutions (pH 4.01
and pH 9.18) following the instruction of manual. Buffer tablets were dissolving in
100 mL of double distilled water (DDW) for preparation of standard buffer solutions.

Procedure:
1. Take 5-10 gm sample in a beaker and add 50 ml D/W.
2. Put the beaker on shaker for 30 min.

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2) Electrical Conductivity (EC)

Principle:
The amount of salt ( soluble) ions in soil sample can be measured by EC (electrical
conductivity). The electrical resistance of soil: water (1:5) suspension measured by
conductivity electrode (Digital pH, conductivity & temperature meter, 181).
Reagents:
Reference solution of 0.01 M KCl
Procedure:
1. Prepared 1:5 (sample: water gm/mL) sample put into a bottle and adding 50 mL
distilled water. Then shake it on shaker at 15 rpm for 1 h to dissolve soluble salts.
2. Calibrate the conductivity meter with standard procedure using the 0.01 M KCl
solution to gain the cell constant and rinse the cell after analysis. The value show
on conductivity meter display and note.

3) Organic Carbon (Walkley and Black Method)

Reagents:
Potassium Dichromate (K2Cr2O7) (1 N) Solution; Standard ferrous ammonium
sulphate (FAS) titrant, approximately 0.5 N; Diphenylamine indicator solution; Conc.
Sulphuric acid, H2SO4 (Sp. gravity 1.84, not less than 96 %) containing 1.25 % silver
sulfate; Ortho-phosphoric acid
Procedure:

1. The oven-dried sample is ground and completely passed through 0.2 mm sieve
(80-mesh) and 0.5 gm is taken in dry 500 mL conical flask (Corning/Pyrex).
2. Add accurately 10 mL of 1 N K2Cr2O7 in the conical flask and swirled the flask
gently to dissolve the sample in the dichromate solution. The flask is set aside on
asbestos sheet.
3. Add 20 mL conc. H2SO4 (containing 1.25% Ag2SO4) very suspiciously from a
measuring cylinder. Swirled two to three times. The flask is allowed to stand for
30 minutes. Protect the flask.
4. Add 200 mL of distilled water and 10 mL of ortho-phosphoric acid to get a
sharper end point of the titration.

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5. After the addition of 1 mL of diphenylamine indicator, the content is titrated with

ferrous ammonium sulfate (Fe(NH4)2(SO4)2.6H2O) solution till the color flashes
from blue-violet to green.
6. Concurrently a blank is run without adding sample.

Calculation:
Organic Carbon, in %= 10(B−T) × Normality of K2Cr2O7×0.003×100
B×S
Whereas, B = Volume of ferrous ammonium sulfate required for blank titration in mL
T = Volume of ferrous ammonium sulfate needed for soil sample in mL,
S = Weight of soil sample.
Organic Matter, in %= Organic Carbon (%) × 1.724

4) Ash content
The estimation of ash content was carried out by standard method21. Dry powered of
seaweed obtained in muffle furnace for 4 h at 550 °C and quantified gravimetrically.

 Estimation of Primary macro nutrients

1) Total Nitrogen (Kjeldahl Nitrogen)
Reagents:
Digestion reagents; Phenolphthalein indicator; Mixed indicator; Boric acid solution
(indicating); Sodium hydroxide (NaOH) 6 N solution; Sulphuric acid 0.02 N
Procedure:
Digestion:

1. Cool & add gently 50 mL digestion reagent to distillation flask.

2. Add few glass beads in it, after mixing with suitable ejection apparatus to remove
acid fumes.
3. Boil it up to the volume of 25 to 50 mL and observed white fumes. Then keep it
for 30 min continue to digest it when colors become pale green and transparent.
4. Digested sample let cool and dilute with 300 mL of water.
5. After dilution add phenolphthalein indicator carefully in amount of 0.5 mL and
sufficient amount of NaOH reagent to form pink zone bottom of flask.

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Distillation:
1. The flask connects with steamed-out distillation equipment and swirl flask up to
total mixing. To take 200 mL distillate and use 50 mL boric acid as absorbent
solution.
2. Absorbent solution level below in extend tip of condenser and don't give
temperature access condenser rise above 29°C and proceed distillation during last
1 or 2 min to rinse condenser.
Final ammonia measurement:

1. To titrate ammonia in distillate with standard 0.02 N H2SO4 titrant until indicator
turn pale lavender.
Blank:

1. Carry a blank through all steps of the procedure and apply the necessary
correction to the results.
Calculation:
Norg (mg/L) = (A−B)×N×14000
Sample Vol. in mL

Whereas; A = volume of H2SO4 titrated for sample, in mL,

B = volume of H2SO4 titrated for blank, in mL,
N = normality of standard H2SO4 titrant solution.
Total Kjedahl Nitrogen (mg/L)= NH3+Norg

2) Total Phosphorus (Bray’s Method)

Reagents:
Bray‟s No.1 Reagent; Dickman‟s and Bray‟s Reagent; Hydrochloric acid, 10 N
Hydrochloric acid, 0.025 N; Sulfuric acid, 7 N; Stannous chloride stock solution (40
%); Stannous chloride diluted solution; Stock Phosphorous solution (100 µg P/mL);
Standard Phosphorous solution (2 µg P/mL)
Procedure:
Extraction:
1. Take 5 gm of soil and 50 mL of the Bray‟s reagent in a 100 mL conical flask and
shake for 5 minutes. Sieve it through Whatman No. 42 paper. If filtrate is not
clear, filter it again.

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Color Development and Estimation:

1. Take 5 mL of the soil extract (filtrate) into a 25 mL of volumetric flask to which
add 5 mL of Dickman‟s and Bray‟s reagent.
2. The neck of the flask is washed down and the contents are diluted to about 22 mL.
3. Then 1 mL of the dilute stannous chloride solution is added and volume is made
up to the mark.
4. The strength of the blue color is measured at 660 nm wavelength on
spectrophotometer just after 10 minutes and the concentration of phosphorous is
determined from the standard curve.
5. This is very important to measure intensity within prescribed time as the color
starts fading after some time.
6. With each set of samples, a blank is (without soil) also run.

Development of standard curve for Phosphorous:

1. Preparation of the standard curve with various concentration of phosphorous (1, 2,
3, 4, 5 and 10 mL of 2 µg P/mL phosphorous solution) are taken in 25 mL
volumetric flasks.
2. Prepare the standard concentrations in the range of 0.08 µg P/mL to 0.80 µg
P/mL.
Calculation:
Bray‟s P, in kg/ha= R × VE × 1 × 2.24 × 106
VA WS 106
Whereas; R = µg of phosphorous in aliquot (obtained from curve),
VE = Volume of Extracting solution (50 mL),
VA = Volume of aliquot taken for analysis (5 mL),
Ws= Weight of soil sample taken for extraction, in g.
3) Potassium (Flame photometric method)
Reagents
Deionised distilled water; Stock potassium solution (1.907 gm/L); Intermediate
potassium solution; Standard potassium solution
Procedure
Nitric acid digestion
1. Take (50-100 mL) mixed sample in a beaker and 5 mL concentrate HNO3.
2. Take it on hot plate and evaporate up to volume of 10 to 20 mL.

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3. Add 5 mL conc. HNO3 and 3 cover with a glass plate, heat to gain gentle
refluxing action.
4. Continue heating and adding conc. HNO3 until a light coloured clean solution is
obtained.
5. Add 1-2 mL concentrate HNO3 and warm slightly to dissolve any remaining
residue.
6. Rinse down the walls of the beaker and glass plate with distilled water and then
filter.
7. Transfer the filtrate to l L volumetric flask, cool and makeup the volume.
Calculation
For direct reference to the calibration curve:
mg K/L = (mg K/L in portion) x D
D = dilution ratio = mL sample + mL distilled water
mL sample

 Estimation of secondary macro nutrients

1) Ca (Calcium)
Reagents:
Ammonium Acetate (1 N); Eriochrome black T indicator; Murexide (ammonium
purpurate); Standard EDTA (0.01 N); Ammonium chloride –ammonium hydroxide
buffer solution; Sodium Hydroxide (4 N); Standard Calcium Solution (0.01 N)
Procedure:
Preparation of Ammonium Acetate Extract (NH4OAc Extract) of sample:
1. Weigh 2.0 g of sample into a 250 mL of beaker. Add 40mL of the ammonium
acetate extracting solution and stir the solution with glass rod for few minutes.
Then keep the solution for overnight. Filter the solution through a Whatman No.
42 or Equivalent filter paper.
Estimation of total Ca2+:
1. Take another 50 mL extract of soil or appropriate mL of extract of soil diluted to
50 mL in conical flask.
2. Add 5 mL of 4 N Sodium hydroxide ( NaOH) which will precipitate Magnesium
sulfate (MgSO4) as insoluble product Mg(OH)2.
3. Add pinch of Ammonium purpurate [C8H8N6O6(MX)] indicator.

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4. Run blank in same manner using 50 mL of the ammonium acetate extracting

solution (1 N) instead of the soil extract solution.
5. Note down the volume of standard EDTA titrant used for soil extract as C and for
blank as B2.
Calculation:
Calcium, in mg/kg= (C−B2) × Normality of EDTA × 40080 × 20
Sample Vol., in mL
Whereas; C = Vol. of EDTA titrant used for Soil extract (for Ca2+ determination),
B2 = Vol. of EDTA titrant for Blank (for Ca2+ determination),
20 = Vol. of NH4OAc extracting solution/weight of soil sample (40 mL/2 g).

2) S (Sulphur)
Reagents:
Nitric acid 25% approximate; Acetic-phosphoric acid; Gum-acacia-acetic acid
solution; Barium sulfate seed suspension; Barium chloride crystals; Concentrated
standard sulfate solution (200 µg/mL); Working standard sulfate solution (10 µg/mL)
Procedure:
Phosphate Extractable SO42-:
1. Take 20 gm of air-dried soil in a 250 mL conical flask.
2. Add 100 mL of 500 ppm solution prepared from KH2PO4 (2.195 gm/L) or
Ca(H2PO4)2H2O (1.888 g/L) into the conical flask. Shake for 30 minutes.
3. Filter the suspension through Whatman No. 42 filter paper.
4. Determine the sulfate from suitable amount of aliquot by turbidimetric method.
Estimation:
1. An appropriate aliquot of the extract (not more than 15 mL and containing less
than 120 µg of sulfate sulfur) is taken in a 25 mL volumetric flask to which 2.5
mL of 25% nitric acid and 2 mL of acetic-phosphoric acid are added and diluted
to about 22 mL. The flask is stoppered and shaken.
2. Then 0.5 mL of barium sulfate seed suspension (before use it must be shaken) and
0.2 g of barium chloride crystals are added successively. The flask is stoppered
and inverted three times.
3. After 10 minutes it is inverted 10 times and after 5 minutes another 5 times.
4. Allowing for another 5 minutes, 1 mL of gum acacia-acetic acid solution is put in
and diluted to volume and inverted 3 times and set aside for 1 h 30 minutes.

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5. Then the flask is again inverted 10 times and reading is taken in

spectrophotometer at 440 nm. The concentration of sulfur is found out from the
standard curve.
Development of standard curve for Sulfur as SO42--S:
1. Prepare the working standards of SO42--S by diluting 0.1, 3, 5, 8, 10 and 12 mL of
working standard sulfate solution into 25 mL volumetric flask.
Calculation:
Sulfur, in kg/ha= R × VE × 1 × 2.24 × 106
VA WS 106

Whereas; R = µg of sulfur in aliquot (obtained from curve),

VE = Volume of Extracting solution (100 mL),
VA = Volume of aliquot taken for analysis (5 mL),
Ws= Weight of soil sample taken for extraction, in g.

3) Mg (Magnesium)
Digestion
The sample was digested with perchloric acid-nitric acid (1:4 V/V) and diluted it with
double distilled water (DDW).
Reagents
Deionised water, resistivity > 18.2 Mohm/cm
Metal Stock solution 1000 mg/L Multi std. VHG Lab. USA
Procedure
1. Calibrate using the blank and standard and then analyze of blank and sample
solution.

 Estimation of micro nutrients

Micro nutrients of seaweed liquid fertilizer like B (boron), Cu (copper), Fe (iron), Mo
(molybdenum), Zn (zinc) and Mn (manganese) were analyzed at SICART (procedure
mention above).

 Estimation of growth hormone

Plant growth hormone of auxin, gibberelline and cytokinin were analyzed at
Junagadh Agriculture University, Junagadh.

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1) Auxin
Sample preparation: 1. Each sample was homogenized with 80% ethanol and
incubated overnight at 4◦C. 2. Filter each sample and vacuum evaporation to remove
all traces of ethanol and residue used. 3. The dry residue was dissolved in 0.02 ml of
80% ethanol & measured.
2) Gibberelline (Method describe in auxin estimation)
3) Cytokinin
First 3 steps of sample preparation are same as auxin measurement. The dry
residue was dissolved in 0.02 mL of 80% ethanol & measured. The residue was
diluted with distilled water and acidified with HCl to pH 2.5 and partitioned twice
with peroxide free diethyl ether (ratio of organic to aqueous phases was 1:3). Organic
phase into 1% Sodium bicarbonate (pH 7-8; the ratio of organic to aqueous phase was
3:1 and re-extracted with diethyl ether and measurement.

 In vitro effect of seed priming of all seaweed liquid fertilizer on

seed germination of selected seeds and Paper Towel method
experiment set up
Priming treatment is pre-soaking of seeds in the different solution to induce
early germination.
 The experiments were conducted using certified Solanum melongena L.,
Lycopersicon esculentum Mill. & Capsicum annuum L. seeds collected from
Vegetable Scientific research center, Anand Agriculture University, Anand,
Gujarat and Brassica oleracea var. Capitata L. and Allium cepa L. seeds
collected from Gandhi Agro, Anand.
 50 healthy seeds were sterilized in 0.1% HgCl2 up to 2-5 minutes and washed
several times with distilled water. After sterilizing seeds were immersed in
different concentration such as 1%, 2%, 3%, 4% and 5% of each seaweed
liquid fertilizer and in water (as a control) up to 48 hours at room temperature
(Plate 6). Immersed seeds were removed and dried on filter paper for one
minute for reached up to their original weight of moisture content67.
 The primed seeds were transferred on tissue paper to provide enough space for
seeds germination. The tissue paper was fold two times and put into zip locked
bag carefully for maintaining the moisture content. The paper towel

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experiment was run up to 12 days (Plate 7). The experiment was performed in
triplicate of each treatment during 2015- 16 to 2017- 18.
 The germination of Solanum melongena, Lycopersicon esculentum, Capsicum
annuum L & Brassica oleracea var. Capitata and Allium cepa seeds were
recorded on 3rd and 4th day respectively. After 12 days, percentage
germination, root length, shoot length, seedling length, seed vigour index
(SVI), seed stamina index (SSI), germination index (GI), and mean
germination time (MGT) were measured by described formula as below.
The percentage germination was calculated by formula47:
Percentage Germination= n × 100
N (1)
Whereas, n= number of seeds that were germinated,
N= total number of seed in each experiment

After final day of germination seedling length was measured with scale
Seedling length= Root length+ Shoot length (2)

Seed Vigour Index (SVI) was calculated by formula3:

Seed Vigour Index = Seedling length (cm) × % Germination (3)
Whereas, Seedling length= Root length+ Shoot length (cm)

Seed stamina index (SSI) measured by formula2:

Seed Stamina Index= [GP (RL+SL)]
100 (4)
Whereas, GP= Percentage Germination; RL= Root length; SL= Shoot length

Germination Index (GI) was calculated by formula34:

Germination Index= No. of germination seed +…+ No. of germination seed
Days of first count days of final count (5)

Mean Germination Time (MGT) was calculated by formula94:

Mean Germination Time = ∑Dn
∑n (6)
Whereas, n= number of seeds that were germinated on day D;
D= number of days counted from beginning of germination

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Plate 6

Solanum melongena L. Lycopersicon esculentum Mill.

Capsicum annuum L.

Brassica oleracea var. Capitata L. Allium cepa L.

Soaking of selected vegetable seeds

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Plate 7

Paper towel method of selected vegetable seeds

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 Ex- situ experiment: the effect of seaweeds as organic fertilizer on

selected vegetables seeds
Preparation of pots for germination of seeds
 Soil sample was collected up to depth of 5-10 cm from agricultural farm at
Anand, Gujarat, India. The soil sample were transferred at home and dried to
get its uniformity.
 Weight 2 kg of dry soil and transferred in each pot. The experimental set up of
11 pots for each vegetable seed with potential 4% concentration which
screening by paper towel method and 1 pot for control. Total 55 pots ready for
run of experiment and all experiment in triplicate sets run during 2015-16 to
2017- 18 under natural condition.
 All vegetable healthy 50 seeds were primed in each seaweed liquid fertilizer
and sawing in enough space in each pot. After sawing sprinkle the water for
maintaining the moisture content for healthy germination. Seaweed liquid
fertilizer applied by soil drench method after 7 days and 14 days.
 After 20 days, germination parameters like percentage germination, root
length, shoot length, seedling length, seed vigour index (SVI) and seed
stamina index (SSI) were carried out by formula was already mention in paper
towel method.
 Biochemical parameters such as chlorophyll-a, chlorophyll-b & total
chlorophyll, carotenoid, total soluble sugar and protein were measured after 20
days by accepted procedures of biochemical analysis296.
 The experiment was conducted in Completely Randomized Design (CRD)
having 3 replications of each selected vegetables.

 Soil analysis
Soil analysis of pH, Electrical conductivity & Organic carbon (C); primary
macro nutrients of Total Nitrogen (N), Total Phosphorus (P) & Total Potassium (K);
secondary macro nutrients such as Calcium (Ca), Sulphur (S) and Magnesium (Mg)
and micro nutrients such as Boron (B), Copper (Cu), Iron (Fe), Molybdenum (Mo),
Zinc (Zn) and Manganese (Mn) were analyzed and all methods describe previously).

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 Biochemical parameters
1) Chlorophyll ‘a’, ‘b’ & total Chlorophyll and Carotenoid
Principle
Chlorophyll is an essential constituent of photosynthesis and occurs as green pigments
in all photosynthetic organisms. Chlorophyll is an organic compound macromolecules
generally present in the green parts of the plants which dissolve in the organic
solvents acetones. This dissolved chlorophyll molecules in acetone are measured at
different nm wavelength of light. There levels are altered during various physiological
conditions25, 316, 253.

Reagents
80 % acetone (per chilled); MgCO3 (Magnesium carbonate)
Procedure
1. Grind 0.1 gm of plants material with 80% of acetone, by adding pinch of MgCO3.
2. Make a homogenize paste of plant materials, transfer it in the centrifuge tube and
centrifuge it at 5000 rpm at 4 °C for 5 min.
3. Transfer the supernatant in volumetric flask make up 25% of volume by using 80%
of acetone.
4. Take the OD at 645, 663 and 480 nm.
Calculation

Chlorophyll a, mg/g = 12.7 (A663) – 2.69 (A645) x __V_____

1000 x W

Chlorophyll b, mg/g = 22.9 (A645) − 4.68 (A663) x __V____

1000 x W

Total Chlorophyll, mg/g = 20.2 (A645) + 8.02 (A663) x ___V___

1000 x W

Carotenoid (µg/g) = A480 + (0.114 × A663) – (0.638 × A645)

2) Protein (Lowry method)

Proteins are polymers of amino acids which are connected to each different by way of
peptide bonds (-CO-NH). The properties of proteins are governed by way of the
different “R" groups of the different amino acids174.

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Principle
Protein reacts with Folin - Coicalteu reagent (FOR) to give a blue colored complex.
The color formation is occurring due to the reaction between of alkaline CuSO4 and
protein. The intensity of the blue color is measured by colorimetric method at 660 nm.

Reagents

80% ethanol; 0.1 N NaOH; Lowry solution A; Lowry solution B; Solution „C‟
(alkaline copper solution); Folin-Coicaiteu reagent (FCR); 10 % trichloroacetic acid
(TCA); 1 N NaOH; Stock standard protein solution; Working standard solution

Procedure

1. Grind 0.1 gm of sample by using 80% acetone in mortar and pastel.

2. Transfer the sample in centrifuge tube and centrifuge it at 5000 rpm for 4°C for 5
mints
3. Discard the supernatant take the pallet and make the volume up to 15 mL pure
distilled water
4. Again centrifuge it and discard the supernatant.
5. Take the pallet and add 10 mL of 0.1 N NaOH. Shake it properly and keep it 10
mints at room temperature, shake it gently for 10mints
6. Centrifuge the tube and take the supernatant in measuring cylinder, make up the
volume up to 10 mL with 1 N NaOH. Shake it properly.
7. Add 5 mL of Lowry reagent and keep it for 30 mints at room temperature.
8. Prepare the standards by taking 1.0, 0.8, 0.6, 0.2, and 0.0 as a blank.
9. Add 0.5 mL of Folin reagent as well as in sample tube, shake it properly.
10. Furfural blue color is obtained.
11. Measure the value of OD at 660 nm by using spectrophotometer and calculate the
amount of protein content (in miligram per gram) in sample by using standard
graph.

3) Total soluble sugar (Anthrone Method)

Principle
Quick and suitable method for the determination of Hexoses, aldopentose and
hexurionic acids either free or present in polysaccharides carried out by the anthrone
reagent. Dehydration of carbohydrates done by concentrated H2S04 to form furfural

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condenses with anthrone. It gives blue-green color which is measured colorimetrically

at 630 nm.
Reagent
80% Ethyl alcohol (Ethanol); Anthrone reagent; Standard Glucose solution
Procedure
1. Take 0.1 gm of plant material in mortar and pastel.
2. Grind it with 80% ethanol.
3. Transfer the sample into centrifuge tube and centrifuges it at 5000 rpm to 4°C for 5
mints.
4. Take the supernatant and transfer into measuring cylinder and make up the volume
up to 25 mL with 80% ethanol, shake it properly.
5. Then from that 25 mL transfer 1ml of sample in separate sugar tube.
6. Keep it in water bath until it gets dried up.
7. Prepare the standards with different aliquots of 0.0 to 1.0 mL of working standard.
8. Add 1 mL of distilled water including sample tube, sake it properly.
9. Add 4 mL Anthrone reagent then keep it again in water bath for 8mints.
10. Cool rapidly, dark green color is obtained.
11. Take the OD at 660 nm, draw a standard graph by plotting concentration of the
standard on the X-axis various absorbance on the Y-axis.
12. Calculate by graph of the amount of carbohydrate present in the sample (mg/gm).

4) Moist weight and Dry weight

Dry weight of sample can be expressed by excluding its water. Dry weight content
indicates the nutrient level in feed sample.This is also known to as “Dry Basis”, “Dry
results” or “Moisture-free Basis”. As there is notable variation in the moisture content
of forages, exclusive of the water or expressing the nutrient level on a dry weight
basis removes the dilution effect of the water, thereby providing the essential common
basis for direct comparison of nutrient content across forages17, 18.

Procedure
Dry the dishes in 105ºC oven overnight. Place in desiccators, cool and weight. Handle
dishes with metal weight 3 gm of the sample into a weighed dish. The close dish
placed in oven at 105ºC for overnight, cool it in the desiccators and weigh as quickly
as possible.

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Calculation
Calculating moisture content from moist weight and dry weight
McWb = Wi−Wf ×100
Wi
Mc db= Wi−Wf ×100
Wi
Mc Wb=Moisture content wet basis (%)
Whereas, Wi = Initial weight; Wf =Final weight
Weight loss during drying
Wf = Wi × 100−Mci
100−Mcf
Whereas, Wi = Initial weight (g); Wf =Final weight (g)

 Antioxidant activity- DPPH assay

Antioxidant activity of selected vegetable seeds measured by using stable
radical 2, 2- diphenyl-2- picrylhydrazyl (DPPH)59. This method is based on the
capability of the antioxidant to scavenge the DPPH cation radical.
Procedure
1. The reaction mixture prepared by mixing 0.1 mL of sample extract and 2.5 mL of
DPPH reagent (prepared in methanol).
2. And add methanol to make final volume 3 mL.
3. Incubate it for 30 min in dark condition at room temperature.
4. Discoloration of DPPH was considered aligned with blank at 517 nm.
5. The scavenging activity of reaction mixture measured by using following formula:
% Antioxidant activity= (absorbance at blank – absorbance at test) × 100
(absorbance at blank)

 Enzyme activity
In pot method, after germination 21 days of aging plantlets material with control and
different treatments were ground with a mortar-pestle in 100 mM HEPES-NaOH (pH-
7.5), 5 mM MgCl2 and 1 mM dithiothretol. The ratio of buffer to plant material was
3:1. The prepared extract was filtered throughout two layers of muslin cloth and
clarified by centrifugation at 15,000 gm for 15 min. The supernatant was used for
catalase and peroxidase activity.

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1) Catalase
Principle
The enzyme activity is assayed by titrimetric method and carried out by estimating the
residual H2O2 in the reaction mixture, and it oxidized by potassium permanganate.
(KMnO4).
Reagent
0.1 M Potassium phosphate buffer (pH 7.0); 0.005 M H2O2: Prepare fresh; 0.7 N
H2SO4; 0.01 N KMnO4
Enzyme extraction
1. Grind the sample (1.0 gm) with 0.1 M phosphate buffer, pH 7.0 in chilled mortar
and pestle.
2. Centrifuge at 15,000 gm for 30 min at 4ºC.
3. Use the supernatant as enzyme source.
Procedure (modified 40)
1. Pipette out 3 mL of phosphate buffer, 2 mL of H2O2 and 1 mL of enzyme extract
into a test tube.
2. Incubate at 20ºC for 1 min.
3. After 1 min bring to an end the reaction by adding 10 mL of 0.7 N H2SO4.
4. Titrate the reaction mixture against 0.01 N KMnO4 to find out the residual H2O2
until a faint purple color persists for at least 15secs.
5. Prepare the blank with adding the enzyme extract.
Calculation
Calculate the enzyme activity and specific activity as units/min and as
units/min/mg protein, respectively or per g sample. One units of catalase is defined.

2) Peroxidase
Principle
The enzyme activity is assayed using o-dianisidine (C6H3)2 as an alternative substrate.
Peroxidase (POD) activity can be assayed by measuring oxidized o-dianisidine
(yellow/orange color) spectrophometrically at 430 nm .
Reagents
Potassium phosphate buffer (0.1 M & pH 6.0); freshly prepared o-Dianisidine (1
mg/ml methanol); 0.2 M Hydrogen peroxide (H2O2), 2 N Sulfuric acid (H2SO4)

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Enzyme extraction
1. Homogenize the material in ice-cold phospahte buffer condition by using mortar
pestle means.
2. Filter it with double layer of muslin cloth and then centrifuge filtrate at 16,000
rpm for 20 min at 4 ºC. Use supernatant as an enzyme extract.
Procedure285
1. Take 3.5 mL Phosphate buffer (pH 6.5) in a clean dry cuvette.
2. Add 0.2 mL enzyme extract and 0.1 mL o-dianisidinesolution freshly prepared.
3. Incubate the mixture at 28-30ºC.
4. The cuvette put in to the spectrophotometer and set at 430 nm.
5. Then add 0.2 mL of 0.2 M H2O2 and quickly start the stop watch.
6. Measure the absorbance.
Calculation
Express the specify activity of enzyme as units/min/mg protein or per g weight
of sample considering one unit of enzyme as an increase in OD by 1.0 under standard
conditions.

 In situ experiment: Effect of seaweed liquid fertilizer on selected

vegetable plant growth, biochemical constituent and yield
Preparation of field for plantation
 Soil sample was collected for soil analysis up to depth of 5-10 cm from
agricultural farm at Anand, Gujarat, India.
 For 40 days, each selected vegetable plants sowed in respective plots. All
experiments carried out under normal atmospheric condition in triplicate
manner in the year of 2015-16, 2016-17 and 2017-18.
 Different seaweed liquid fertilizer was applied by two applications of foliar
spray (M1) and soil drench (M2) on field.
 Growth parameters such as number of branches, number of leaves and shoot
length were measured after 20, 40 and 60 day and plant height was measured
after 60 days.
 Potential application of seaweed liquid fertilizer was selected on growth
parameters of number of branches, number of leaves and shoot length.

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 Screening the most potential application of seaweed liquid fertilizer and after
biochemical parameters such as chlorophyll-a, chlorophyll-b & total
chlorophyll, carotenoid, total soluble sugar, protein, wet weight, dry weight, %
moisture, % dry matter and yield of crop were measured after 60 days by
standard methods of biochemical analysis296.

 Field experiment
Solanum melongena L. (Brinjal)
Crop period : Rabi seasons of 2015-16 to 2017-18
Treatments : 10 + 1 (control)
Design : Randomized Block Design (RBD)
Replication :3
Spacing : 60 cm × 60 cm
Plot size : 3.6 m × 3 m
Treatments detail: A1- Ulva lactuca; A2- Ulva reticulata
A3- Gracillaria corticata; A4- Kappaphycus alvarezii
A5- Sargassum johnstonii; A6- Padina pavonica
A1+A2- Ulva lactuca + Ulva reticulata
A3+A4- Gracillaria corticata + Kappaphycus alvarezii
A5+A6- Sargassum johnstonii + Padina pavonica
AM- Mixture of all seaweeds

Lycopersicon esculentum Mill. (Tomato)

Crop period : Rabi seasons of 2015-16 to 2017-18
Treatments : 10 + 1 (control)
Design : Randomized Block Design (RBD)
Replication :3
Spacing : 60 cm × 60 cm
Plot size : 3.6 m × 3 m
Treatments detail: A1- Ulva lactuca; A2- Ulva reticulata
A3- Gracillaria corticata; A4- Kappaphycus alvarezii
A5- Sargassum johnstonii; A6- Padina pavonica
A1+A2- Ulva lactuca + Ulva reticulata

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A3+A4- Gracillaria corticata + Kappaphycus alvarezii

A5+A6- Sargassum johnstonii + Padina pavonica
AM- Mixture of all seaweeds

Capsicum annuum L (Chilli)

Crop period : Rabi seasons of 2015-16 to 2017-18
Treatments : 10 + 1 (control)
Design : Randomized Block Design (RBD)
Replication :3
Spacing : 45 cm × 60 cm
Plot size : 3.6 m × 2.7 m
Treatments detail: A1- Ulva lactuca; A2- Ulva reticulata
A3- Gracillaria corticata; A4- Kappaphycus alvarezii
A5- Sargassum johnstonii; A6- Padina pavonica
A1+A2- Ulva lactuca + Ulva reticulata
A3+A4- Gracillaria corticata + Kappaphycus alvarezii
A5+A6- Sargassum johnstonii + Padina pavonica
AM- Mixture of all seaweeds
Brassica oleracea var. Capitata L (Cabbage)
Crop period : Rabi seasons of 2015-16 to 2017-18
Treatments : 10 + 1 (control)
Design : Randomized Block Design (RBD)
Replication :3
Spacing : 50 cm × 50 cm
Plot size :4m×3m
Treatments detail: A1- Ulva lactuca; A2- Ulva reticulata
A3- Gracillaria corticata; A4- Kappaphycus alvarezii
A5- Sargassum johnstonii; A6- Padina pavonica
A1+A2- Ulva lactuca + Ulva reticulata
A3+A4- Gracillaria corticata + Kappaphycus alvarezii
A5+A6- Sargassum johnstonii + Padina pavonica
AM- Mixture of all seaweeds

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Allium cepa L (Onion)

Crop period : Rabi seasons of 2015-16 to 2017-18
Treatments : 10 + 1 (control)
Design : Randomized Block Design (RBD)
Replication :3
Spacing : 15 cm × 10 cm
Plot size : 1.5 m × 1.5 m
Treatments detail: A1- Ulva lactuca; A2- Ulva reticulata
A3- Gracillaria corticata; A4- Kappaphycus alvarezii
A5- Sargassum johnstonii; A6- Padina pavonica
A1+A2- Ulva lactuca + Ulva reticulata
A3+A4- Gracillaria corticata + Kappaphycus alvarezii
A5+A6- Sargassum johnstonii + Padina pavonica
AM- Mixture of all seaweeds

 Effect of seaweed liquid fertilizer on vegetable nutrient content

1) Ascorbic acid (Vitamin-C)
Principle
The color of DCPIP dye (2, 6- dichlorophenol indophenols) decrease by Ascorbic acid
to less leuco base. The ascorbic acid gets oxidized to dehydroascorbicacid (C6H6O6).
Dye color blue converts in to pink color in acidic condition. The facility of titrating
medium served by Oxalic acid.
Reagents
4% Oxalic acid; Dye Solution; Standard stock solution; Working standard solution
Procedure
1. Pipette out 5 mL of the working standard solution into a 100 mL conical flask.
2. Add 10 mL of 4% oxalic acid and titrate against the dye (V1 mL). At the end
point pink color develop which persists for a few minutes. The amount of the dye
consumed is equal to the quantity of ascorbic acid.
3. Sample of extract (0.5-5 gm depending on the sample) in 4% Oxalic acid and then
100 mL volume make up and centrifuge (Plate 8).
4. Pipette out 5 mL of supernatant, add 10 mL of 4% oxalic acid and titrate against
the dye (V2 mL).

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Calculation:
Ascorbic acid mg/100g= 0.5 mg × V2 × 100 mL × 100
V1 mL 15 mL wt. of the sample

2) Protein (Method already describe in Ex-situ experiment)

3) Total Soluble Sugar (Method already describe in Ex-situ experiment)

 Statistical analysis

The laboratory experiments were conducted with three repetition and ten treatments.
Data were analyzed by using analysis of variance (ANOVA) following completely
randomized block design281. Differences were measured significant at 5% level of
probability.

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Plate 8

Extract of vegetable which treated with different seaweed liquid fertilizer

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