Biomass Conversion and Biorefinery

https://doi.org/10.1007/s13399-021-01881-4

ORIGINAL ARTICLE

Comparative study of biofertilizer/biostimulant from seaweeds

and seagrass in Abelmoschus esculentus crop
Durai Murugan Muniswami1 · Saranya Chinnadurai1 · M. Sachin1 · H. Jithin1 · K. Ajithkumar1 · G. Surya Narayanan1 ·
B. S. Gopinath1 · A. Ahamed Rasheeq2 · B. Antrose Preethi3 · R. Dineshkumar1

Received: 1 July 2021 / Revised: 11 August 2021 / Accepted: 14 August 2021

© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2021

Abstract
This study was conducted to evaluate seaweeds/seagrass as a biofertilizer/biostimulant in plant growth and yield. The surplus
use of commercial chemical fertilizer and pesticides has an adverse effect on water and soil quality. The consequences lead
to an indirect toxic effect on humans. Among different sources, marine seaweeds/seagrass is an alternate source for organic
fertilizers to enhance and to restore soil fertility. We aimed to develop organic fertilizer/biostimulant from seaweeds (Sargas-
sum, Turbinaria ornata (TO), Halimeda microloba (HM)) and seagrass. In our experiment, soil and irrigation water quality
were analyzed before lady’s finger (Abelmoschus esculentus) seeds were sown for germination. The seaweeds/seagrass macro-
and micronutrients as well as phytohormones (salicylic acid (SA), abscisic acid (ABA), jasmonic acid (JA), indole-3-acetic
acid (IAA), zeatin (ZE), and gibberellic acid (GA)) were analyzed by calorimetry and LC–MS/MS respectively. The plants
were grown in soil supplemented with 25%, 50%, 75%, and 100% of seaweeds/seagrass by two methods (soil drench (SD)
and foliar spray (FS)) of application, whereas the control group was without any treatment. The plant growth, chlorophyll,
and yield were analyzed. Silt clay loamy soil exhibits water percolation of 3.0 min and water holding capacity of 18%. Water
suitable for irrigation contains ­HCO3, Cl, ­SO4, Mg, and Ca. Nitrogen content (402 mg/L) and phosphorus (60 mg/L) were
increased in TO than those in other seaweeds. Similarly, potassium level (35.4 mg/L), calcium content (86.1 mg/L), magne-
sium (36 mg/L), and amino acid (102 mg/L) were found more in seagrass. SA, ABA, and JA elicit more in Sargassum than
the other sources, with a lesser amount of GA and ZE. Seagrass was enriched in IAA, ZE, GA, JA, SA, and ABA, whereas
GA was found more in HM with lesser amounts of SA, ABA, ZE, and JA. SD application of TO shows increased number of
flowers, number of pods, and length and weight of pods as compared to those in FS. SD application of HM resulted in increase
in growth and yield. FS treatment of Sargassum elicits enhanced growth and number of pods, but length and weight of pods
were increased in SD. FS application of seagrass has shown to increase the number of flowers and pods as compared to SD,
whereas length and weight of pods were significantly increased in SD than those in FS. However, seagrass elicits the highest
in yield than the seaweeds. Seaweeds/seagrass is an alternate source of organic fertilizer/biostimulant for organic farming.

Keywords  Seaweeds · Seagrass · Biofertilizer · Biostimulant · Lady’s finger

1 Introduction

Agricultural products are the minimum essential for humans

* R. Dineshkumar and livestock. There is a huge demand for commercial chemical
dineshram625@gmail.com
fertilizers (nitrogen (N), phosphorus (P), and potassium (K))
1
Department of Microbiology, Karpagam Academy due to increase in population and urbanization. The overuse
for Higher Education, Pollachi Main Road, Eachanari, of commercial N-fertilizer, results in reduced soil quality
Coimbatore, Tamil Nadu 641021, India and associated toxicity to living beings [1]. The utilization of
2
College of Fisheries, Karnataka Veterinary Animal organic fertilizer from marine source is one of the possible
and Fisheries Sciences University, Mangalore, Karnataka, approaches to improving soil organisms and soil content,
India
and restoring essential plant nutrients to the soil [2–4].
3
College of  Fisheries, Central Agricultural University, Although seaweeds could potentially benefit plant growth
Imphal 750 004, India

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 Biomass Conversion and Biorefinery

and yield by providing macronutrients, including nitrogen 100 mL of water. Samples were boiled for 15 min and then fil-
(N), potassium (K), and phosphorus (P). Earlier studies have tered using Whatman paper. Sample extracts were collected and
shown that the beneficial effects of seaweed extracts are not analyzed for six phytohormones, GA, ABA, ZE, IAA, JA, and SA.
due to macronutrients, but may be due to the presence of
other constituents in them that might play a role in biological 2.1.3 LC–MS/MS conditions
activity even at low concentrations [5, 6]. The other compounds
involved are unknown; possibly, several plant growth promoters LC–MS/MS analysis was conducted using the LC sys-
work synergistically [7–9]. Rapid growth rate in plants may tem coupled to a triple-quadrupole mass spectrometer
be due to the presence of growth regulators. Like higher (LC–MS 8040, Shimadzu, Japan). The sample separation
plants, bacteria, and fungi, evidence suggested that algae was achieved on an C18 (150 × 4.6 mm) column. The flow
also synthesize plant growth regulators gibberellic acid, rate was set to 0.3 mL ­min−1 and the temperature of the
abscisic acid, auxin, and cytokinin [10]. Seaweeds are rich column was set at 35 °C. The acquired mass spectra are
sources of plant growth-promoting substances such as kinetin, on both negative and positive modes using electrospray
gibberellins, zeatin, auxin, cytokinins [11], metabolic enhancers ionization and quantification of all analytes was carried
[12], micro- and macroelements [13], vitamins, and amino out in MRM mode. The other operating parameters were as
acids, and their use in crop cultivation shows beneficial results follows: nebulizer gas flow, 3 mL ­min−1; desolvation line
in seed germination, plant growth, and yield [14]. The seagrass (DL) temperature, 250 °C; heat block temperature, 400 °C;
form a fundamental part of the marine ecosystem and they collision-induced dissociation (CID) gas, 230 kPa.
nurture the soil upon application for crop yield. The bioactive
substance from seagrass extract used a biostimulant for the plant 2.2 Elemental analysis
growth in Lycopersicon esculentum L. [53]. The current study
aimed to evaluate the seaweeds (Sargassum, Turbinaria ornata, The total protein (Lowry et al., 1951) [40], carbohydrate con-
and Halimeda microloba) and seagrass as a biofertilizer/ tent (Dubois et al. 1956) [41], lipid content (Bligh and Dyer
biostimulant in Abelmoschus esculentus plant growth and yield. 1959) [42], the fiber content were analyzed using the AOAC
The phytohormones namely salicylic acid (SA), abscisic acid enzymatic gravimetric method (AOAC, 1990); the mineral
(ABA), jasmonic acid (JA), indole-3-acetic acid (IAA), zeatin content such as nitrogen, potassium, phosphorus, iron, man-
(ZE), and gibberellic acid (GA) were screened in seaweeds (T. ganese, and zinc was analyzed by inductively coupled plasma
ornata, Sargassum, and H. microbola) and seagrass. In addition atomic emission spectrometry (Perkin-Elmer Life and Ana-
to hormones, macro- and micronutrients were also analyzed. lytical Sciences, USA) (Marts and Meloan, 1982) [43].

2.3 Soil characteristics
2 Material and methods
Soil was collected at the depth of 15 cm from agricultural
2.1 Sample collection lands of Eachanari, Coimbatore, Tamil Nadu, for crop culti-
vation. Soil profile (pH, EC, water percolation rate, moisture
Seaweeds and seagrass samples were collected from the Gulf of content, water holding capacity, capillary action, N, P, K,
Mannur, at the southeast coast of India. The collected samples Zn, Fe, Cu, and Mn) by following the standard methodology
were transported and authentication was done by the taxono- [38, 39] was screened in Tamil Nadu Soil Fertility Centre,
mist Dr. P. Sampathkumar, CAS in Marine Biology, Faculty of Coimbatore, and each pot was filled with 5 kg of soil.
Marine Sciences, Annamalai University, Tamil Nadu, India.
2.4 Water quality parameters
2.1.1 Chemicals
The water used for irrigation was analyzed for different
Methanol and water of HPLC grade were obtained from parameters such as pH (EC), ­HCO3,CO3, Cl, ­SO4, K,Na,
Sigma. SA, GA, ABA, ZE, IAA, and JA were purchased Ca, Mg, RSC, SAR, and Mg:Ca by following the standard
from Sigma-Aldrich. LC–MS/MS experiment was done in methodology (APHA 1992) [37] done in Tamil Nadu Soil
Tamil Nadu Agriculture University, Coimbatore. Research Centre, Coimbatore.

2.1.2 Extraction 2.5 Lady’s finger plant cultivation

The harvested plants were shadow dried and finely ground into The experiment was conducted at the Karpagam Academy
powder with a mortar and pestle. Each powder sample was then of Higher Education, Eachanari, Coimbatore-21, Tamil
weighed (100 g) and transferred into a conical flask containing Nadu, India, during the period of 2019–2020 to evaluate the

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Biomass Conversion and Biorefinery

potential of seaweeds (Sargassum, T. ornata, and H. micro- 2.8 Pot cultivation

loba) and seagrass as biofertilizer/biostimulant in lady’s
finger (Abelmoschus esculentus) cultivation. The seeds were planted in 30-cm diameter pots. In each pot,
five seeds were sowed, watered, and maintained in natu-
2.6 Soil drench method ral environmental conditions. The growth parameters were
recorded at early and flowering stages of plants. Finally, the
The different concentrations, that is 25% (1.25  g), 50% yield attributes were measured.
(2.5 g), 75% (3.75 g), and 100% (5 g), in powder form of
Sargassum, T. ornata, and H. microloba and seagrass were 2.9 Estimation of chlorophyll
applied by soil drench treatment methods as a biofertilizer.
The chlorophyll a and b and total chlorophyll were meas-
Biofertilizers/treatment Soil drench treatment (SD) Control
ured at the early stage by using the standard methodology
method/concentration
25% 50% 75% 100% of Holden (1965) [51].
Sargassum (SAR) 1.25 g 2.5 g 3.75 g 5 g -
Turbinaria ornata (TO) 1.25 g 2.5 g 3.75 g 5 g - 2.10 Statistical analysis
Halimeda microloba (HM) 1.25 g 2.5 g 3.75 g 5 g -
Seagrass (SG) 1.25 g 2.5 g 3.75 g 5 g - One-way ANOVA LSD was performed using SPSS version
16 for comparison of different groups. The values repre-
sented means and standard deviations (mean ± SD). p < 0.05
2.7 Foliar spray method was considered statistically significant.

Seaweeds/seagrass powder (1 kg) was boiled with distilled water

(1 L) and then filtered. The filtrate was considered as 100% 3 Results
concentration of seaweed liquid extract and from this different
concentrations of 25%, 50%, 75%, and 100% were prepared by The collected specimen was authenticated as seaweeds
mixing with distilled water. The different concentrations were (Sargassum, Turbinaria ornata, Halimeda microloba) and
used by foliar spray method twice weekly. In addition to sea- seagrass (Fig. 1).
weed/seagrass, two commercial liquid spray products were used
to compare the effect—(i) Allwin Gold Super, SDS Ramcides 3.1 Detection of phytohormones in liquid extract
Crop Science Private Ltd; (ii) organic foliar spray, FIESTA-FS,
India (marine amino acids, poly-d glucosamine) —and in another Liquid extract was analyzed by LC–MS-MS for detection
group, commercial chemical micronutrients were treated with dif- of GA, ABA, ZE, IAA, JA, and SA. The compound cor-
ferent doses of 25%, 50%, 75%, and 100%. Control group without responding to mass spectra m/z of 137.20, 174.10, 209.30,
any treatment.

Biostimulant/treatment Foliar spray treatment (FS) Control

method/concentration
25% 50% 75% 100%

Sargassum extract (SAR) 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
distilled water distilled water distilled water
Turbinaria ornata (TO) extract 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
distilled water distilled water distilled water

Halimeda microloba (HM) 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
extract distilled water distilled water distilled water
Seagrass (SG) extract 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
distilled water distilled water distilled water
Commercial fertilizer-I (Com- 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
I) distilled water distilled water distilled water
Commercial fertilizer-II (Com- 25 ml extract + 75 ml 50 ml extract + 50 ml 75 ml extract + 25 ml 100 ml extract -
II) distilled water distilled water distilled water
Micronutrient (MN) 25 ml + 75 ml distilled 50 ml + 50 ml distilled 75 ml + 25 ml distilled 100 ml -
water water water

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 Biomass Conversion and Biorefinery

218.30, 263.30, and 345.20 in LC–MS/MS analysis was negligible levels of GA, JA, and SA were found in
confirmed as SA, GA, ABA, ZE, IAA, and JA respectively the TO. The phytohormonal levels in the seaweeds/
as it is matched with the mass of the standard (Figs. 2, 3, 4). seagrass were shown in increasing order: salicylic
On screening, seagrass elicits all six phytohor- acid (SA): HM < TO < SG < SAR, gibberellic acid
mones (SA, GA, ABA, ZE, IAA, and JA) in our (GA): TO < SAR < SG < HM, abscisic acid (ABA):
study. Similarly, all five hor mones are present in SG < HM < SAR, Zeatin (ZE): HM < SAR < SG,
Sargassum, except IAA. High amounts of gibber- indole-3-acetic acid (IAA): SG, jasmonic acid (JA):
ellic acid present in HM, but lesser expressions TO < HM < SG < SAR (Tables 1, 2, 3, 4, 5).
of SA, ABA, and JA were noticed in HM. The

Fig. 1  Collected marine source

samples were a Turbina-
ria ornata, b Sargassum, c
seagrass, and d Halimeda
microloba

Fig. 2  Multiple reaction moni-

toring (MRM) mass electro-
phorograms of six phytohor-
mone standards. SA, salicylic
acid; GA, gibberellic acid;
ABA, abscisic acid; ZE, zeatin;
IAA, indole-3-acetic acid; JA,
jasmonic acid

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Biomass Conversion and Biorefinery

Fig. 3  Typical MRM chromatograms of working solution of standard plant hormones. SA, salicylic acid; GA, gibberellic acid; ABA, abscisic
acid; ZE, zeatin; IAA, indole-3-acetic acid; JA, jasmonic acid

3.2 Soil physico‑chemical characterization macronutrients were also analyzed. The phosphorus content
before experiment was 5.9 ppm, nitrogen 100 ppm, and potassium 409 ppm.
The micronutrients like iron, zinc, manganese, and cop-
Physical and chemical analysis of the soil was tested prior per were also analyzed as 5.1 ppm, 0.8 ppm, 2.1 ppm, and
to the plant growth experiments. The soil texture grade was 1.1 ppm respectively (Table 6).
found to be silt clay loam. The clay content was 41.1%, silt
was 37.0%, and sand was found to be 21.1%. The physical 3.3 Water quality parameters
parameters like pH, water percolation rate, moisture content,
capillary action, water holding capacity, etc., were observed The water quality parameters were analyzed before the
as part of the analysis. The pH was 7.30; EC was 0.42; mois- experiment; pH of the water was found to be 7.8 mEq/L,
ture content was analyzed to be 3.22. The percolation rate sodium 0.5  mEq/L, potassium 0.3  mEq/L, calcium
was 3.0 min, water holding capacity 18%, and capillary 1.6  mEq/L, magnesium 2.6  mEq/L, SAR 0.403  mEq/L,
action 3.03 min. The chemical parameters like micro- and

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 Biomass Conversion and Biorefinery

Table 2  Multiple-reaction monitoring mode (MRM) for the analysis

of plant hormones in seagrass (SG)
S. no Hormone Rt (min) Area Ion (m/z)

1 Salicylic acid (SA) 1.869 74,435 137.20 > 93.15

2 Gibberellic acid (GA) 1.791 6205 174.10 > 130.05
3 Abscisic acid (ABA) 1.847 1141 209.30 > 59.10
4 Zeatin (ZE) 2.060 51,246 218.30 > 134.20
5 Indole-3-acetic acid 1.922 2406 263.30 > 153.15
(IAA)
6 Jasmonic acid (JA) 1.825 858 345.20 > 143.25

Table 3  Multiple-reaction monitoring mode (MRM) for the analysis

of plant hormones in Turbinaria ornata (TO)
S. no Hormone Rt (min) Area Ion (m/z)

1 Salicylic acid (SA) 1.969 27,302 137.20 > 93.15

2 Gibberellic acid (GA) 1.923 3406 174.10 > 130.05
3 Abscisic acid (ABA)
4 Zeatin (ZE)
5 Indole-3-acetic acid
(IAA)
6 Jasmonic acid (JA) 1.881 506 345.20 > 143.25

Table 4  Multiple-reaction monitoring mode (MRM) for the analysis

of plant hormones in Sargassum 
S. no Hormone Rt (min) Area Ion (m/z)

1 Salicylic acid (SA) 2.005 129,383 137.20 > 93.15

2 Gibberellic acid (GA) 1.975 5577 174.10 > 130.05
3 Abscisic acid (ABA) 3.754 4916 209.30 > 59.10
4 Zeatin (ZE) 2.142 1402 218.30 > 134.20
5 Indole-3-acetic acid
(IAA)
Fig. 4  Typical MRM chromatograms of gibberellic acid (GA), absci- 6 Jasmonic acid (JA) 1.773 1092 345.20 > 143.25
sic acid (ABA), zeatin (ZE), indole-3-acetic acid (IAA) in the extract
of seagrass, and jasmonic acid (JA) in Sargassum extract

Table 1  Multiple-reaction monitoring mode (MRM) for the analysis Table 5  Multiple-reaction monitoring mode (MRM) for the analysis
of plant hormones in Halimeda microloba (HM) of plant hormones in standard
S. no Hormone Rt (min) Area Ion (m/z) S. no Hormone Rt (min) Area Ion (m/z)

1 Salicylic acid (SA) 1.753 20,966 137.20 > 93.15 1 Salicylic acid (SA) 2.246 8,746,489 137.20 > 93.15
2 Gibberellic acid (GA) 1.768 9498 174.10 > 130.05 2 Gibberellic acid (GA) 2.432 76,719 174.10 > 130.05
3 Abscisic acid (ABA) 1.971 2346 209.30 > 59.10 3 Abscisic acid (ABA) 4.689 1,211,534 209.30 > 59.10
4 Zeatin (ZE) 2.081 955 218.30 > 134.20 4 Zeatin (ZE) 2.008 1,595,234 218.30 > 134.20
5 Indole-3-acetic acid 5 Indole-3-acetic acid 3.250 1,826,049 263.30 > 153.15
(IAA) (IAA)
6 Jasmonic acid (JA) 1.789 771 345.20 > 143.25 6 Jasmonic acid (JA) 2.314 471,427 345.20 > 143.25

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Biomass Conversion and Biorefinery

chloride 3.1  mEq/L, sulfate 0.6  mEq/L, bicarbonate Table 7  Quality of water used for irrigation
1.1 mEq/L, magnesium:calcium ratio 1:57 mEq/L respec- S. no Parameters Value (Ml)
tively (Table 7).
1 pH 7.8
3.4 Biochemical nutrient and mineral composition 2 (E.C.) ­dsm−1 0.51
analyses from Halimeda microloba liquid 3 CO3 –
extract 4 HCO3 1.1
5 Cl 3.1
The amino acid content was found to be 69 mg/L and pH 6 SO4 0.6
was 8.0. The mineral content was also determined where the 7 Na 0.5
nitrogen content present is 188 mg/L, phosphorus 53 mg/L, 8 K 0.3
potassium content 14.6 mg/L, calcium 70 mg/L, iron con- 9 Ca 1.6
tent 0.8 mg/L, magnesium 31 mg/L, zinc 1.6 mg/L, boron 10 Mg 2.6
0.6  mg/L, copper 0.78  mg/L, sodium 102  mg/L, sulfur 11 RSC –
4.0 mg/L, and chloride content 89.5 mg/L (Fig. 5). 12 SAR 0.403
13 Mg:Ca 1.57

3.5 Biochemical nutrient and mineral composition

analysis from Turbinaria ornata liquid extract
boron 0.3 mg/L, copper 1.2 mg/L, sodium 102 mg/L, sulfur
The amino acid content was found to be 98 mg/L and pH 4.6 mg/L, and chloride content 56.5 mg/L (Fig. 5).
was 7.8. The mineral content was also determined where
the nitrogen content present is 402  mg/L, phosphorus 3.6 Biochemical nutrient and mineral composition
60 mg/L, potassium content 33.4 mg/L, calcium 77 mg/L, analyses from Sargassum liquid extract
iron content 3.0 mg/L, magnesium 25 mg/L, zinc 2.7 mg/L,
The amino acid content was found to be 80 mg/L and pH
was 8.2. The mineral content was also determined where
the nitrogen content present was 230 mg/L, phosphorus
Table 6  Physical and chemical analyses of the soil before experi-
47 mg/L, potassium content 18.5 mg/L, calcium 83 mg/L,
ments
iron content 1.2 mg/L, magnesium 36 mg/L, zinc 2.4 mg/L,
Characters Value boron 0.5 mg/L, copper 0.28 mg/L, sodium 95 mg/L, sulfur
Particle size distribution 3.0 mg/L, and chloride content 74.3 mg/L (Fig. 5).
Clay 41.1
Silt 37.0 3.7 Biochemical nutrient and mineral composition
Sand 21.1 analyses from seagrass liquid extract
Texture grade Silt clay loam
Physical parameters The amino acid content was found to be 102 mg/L and
pH (1:2.5 suspension) 7.30 pH was 6.2. The mineral content was also determined
EC (1:2.5) mmohs/cm 0.42 where the nitrogen content present was 375 mg/L, phos-
Moisture content 3.22 phorus 27 mg/L, potassium content 35.4 mg/L, calcium
Water percolation rate 3.0 (min) 86.1 mg/L, iron content 4.2 mg/L, magnesium 30.7 mg/L,
Water holding capacity 18% zinc 3.8 mg/L, boron 0.1 mg/L, copper 1.7 mg/L, sodium
Capillary action 3.03 (min) 81 mg/L, sulfur 2.7 mg/L, and chloride content 56.2 mg/L
Chemical parameters (Fig. 5).
Available macronutrients
N ppm 100 3.8 Early stage of growth assessment
P ppm 5.9
K ppm 409 The number of leaves was found to be increased in treated
Available micronutrients plants as compared to control in both the methods of Tur-
Zn ppm 0.8 binaria ornata. The number of branches was higher in the
Fe ppm 5.1 foliar spray of T. ornata treatment. There is a significant
Cu ppm 1.1 difference in plant height of T. ornata-treated plants as com-
Mn ppm 2.1 pared to control. Soil drench treatment of 100% TO shows

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 Biomass Conversion and Biorefinery

Fig. 5  Nutrient analysis in
seaweed/seagrass

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Biomass Conversion and Biorefinery

the highest in plant height than the other concentrations in height in all the treatment doses as compared to control.
(Fig. 6). The treatment of Sargassum by soil drench showed enhanced
Soil drench application of Halimeda microloba (HM) plant growth in height as well as in the number of leaves,
elicits an increased number of leaves than that of foliar spray. whereas there were more branches in foliar spray of Sar-
Similarly, soil drench HM demonstrates significant increase gassum. Seagrass treatment shows statistical significance in

Fig. 6  Early stage of growth analysis

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 Biomass Conversion and Biorefinery

plant height in both the methods of application as compared seagrass treatment, the foliar spray method demonstrates the
to the control group (Fig. 6). higher number of flowers and pods, whereas the soil drench
In foliar spray, the low dose of 25% elicits significance in method shows increase in pod length and weight (Fig. 10).
plant height as compared to control, except HM, Sargassum, In 25% of foliar spray, length and weight of pods were sta-
and seagrass. The 75% foliar spray treatment shows increase tistically significant in all treatment groups as compared to
in height as compared to the untreated group. There is not control, but lesser than the commercial spray. In the higher
much difference in the number of leaves among the treat- dose (100%) of foliar spray, all treatment groups tend to give
ment groups (Fig. 7). more yield than control, and seagrass and TO exhibit similar
yields with those of the commercial product (Fig. 11).
In foliar spray treatment, the yield (length of pod)
3.9 Chlorophyll analysis assessment was shown in increasing order: con-
trol < MN < HM < TO < SAR < Com-II < Com-I < SG. In
Chlorophyll synthesis requires many elements such as foliar spray treatment, the yield (weight of pod) assessment
nitrogen (N) and phosphorus (P). Chlorophyll “a” ranges was shown in increasing order: control < MN < HM < Com-
from 2.0 to 4.2 mg/mL in soil drench method of treatment, II < Com-I < SAR < TO < SG (Fig. 11). Overall, the efficacy
whereas in foliar spray, it ranges from 2.1 to 4.3 mg/mL in of seaweeds/seagrass was shown in increasing order: con-
the lady’s finger plant. Chlorophyll “b” ranges from 0.31 to trol < TO < HM < SAR < SG (Figs. 10, 11 and 12).
1.2 mg/mL in the soil drench method, and 0.26 to 1.27 mg/
mL in the foliar spray method. Both the methods of applica-
tion showed increase in chlorophyll content as compared to 4 Discussion
control. Overall, the chlorophyll content is shown in increas-
ing order: HM < SG < SAR < TO (Fig. 12). The practice of using synthetic fertilizer, pesticides, and
insecticides spoils the soil ecosystem, which could be unsuit-
able for crop cultivation. Soil nutrients will get depleted due
3.10 Flowering stage of growth assessment to intensive cropping. In addition, unsuitable soil conditions,
like salinity, calcium carbonate content, high or low pH [15,
Among the soil drench and foliar spray methods, foliar 16], and the antagonism between micro- and macronutrients
spray of Turbinaria ornata treatment shows increase in [17], can also affect the availability of nutrients. Apart from
plant height and number of branches. A 100% Halimeda micronutrients, macronutrients are also the basic essential
microloba treatment resulted in a higher number of leaves for the growth of all crops [18]. They are involved as co-
and branches and height in either treatment method. Among enzymes and in the redox systems for different metabolic
the different doses, 100% treatment of Sargassum shows processes in plants [19, 20]. Thus, cellular activities are
the highest number of leaves and branches and plant height. affected due to micronutrient deficiency, which ultimately
Foliar spray of Sargassum demonstrates to be better than soil leads to abnormal growth of plants and low yield with poor
drench in terms of plant growth. The foliar spray of seagrass quality [15, 21].
exhibits a higher number of leaves and branches and plant Recently, the use of natural plant biostimulant in crops
height as compared to soil drench as well as control group is an innovative approach. Biofertilizers/biostimulants are
(Fig. 8). Among the foliar spray, a 100% treatment dose elic- an eco-friendly, positive influence on soil sustainability and
its statistical significance in plant height with the treatments increase plant productivity. Algae are grouped into microal-
of seagrass, Sargassum, and T. ornata (Fig. 9). gae and macroalgae. Some of the microalgae commercially
available are Chlorella species, Isochrysis species, Chaetoc-
3.11 Yield assessment eros species, Dunaliella species, and Arthrospira species,
which are applied in different sectors such as pharmaceuti-
Turbinaria ornata (TO) treatment elicits a significant cals, agriculture, and food industries [22, 44–47]. Macroal-
increase in the number of flowers, number of pod, and pod gae or seaweeds are divided into three categories based on
length and weight as compared to control, but the soil drench their pigmentation: Rhodophyta (red), Phaeophyta(brown),
method enhances the length of the pod and weight of pods. and Chlorophyta (green) [23]. Marine-sourced seaweeds are
The soil drench method of HM treatment shows the better macro-algae found in shallow coastal regions with enormous
number of flowers, number of pods, and weight and length nutrient content in it [36].
of pods than the foliar spray. The number of flowers and The biochemical composition of seaweed extract is a
length of pods were increased in the soil drench method of complex which contains minerals, polysaccharides, fats,
Sargassum treatment than that in foliar spray, but both the vitamins, oils, acids, antioxidants, hormones, and pig-
Sargassum treatments were better than the control group. In ments [23–25]. Recently, seaweed extract was widely used

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Biomass Conversion and Biorefinery

Fig. 7  Early stage of growth analysis in foliar spray method of application

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 Biomass Conversion and Biorefinery

Fig. 8  Growth analysis during flowering stage

as a plant biostimulant, any substance applied to plants literature [29, 52]. Veeranan U et al. demonstrated the
with the target to enhance nutrition efficiency, crop qual- biofertilizer activity of Sargassum wightii, and Turbinaria
ity traits, and/or abiotic stress tolerance, regardless of its ornata in Ocimum sanctum [30].
nutrient content [22]. Seaweed extract can be applied to The outcomes of green algae C. vulgaris liquid extract as
plants and/or soil as a foliar spray. They act positively on a foliar fertilizer on composition status of nutrients, and the
remediation, soil retention, and soil microflora and also are yield and growth of wheat crop were analyzed, which were
sources of many nutrients/hormones [26]. compared to those of a synthetic foliar fertilizer [31, 48].
Seaweed extract is used as a fertilizer in different forms The 50% v/v administration of microalgal cell extract was
such as seaweed liquid fertilizer (SLF) and powder form found to be the best treatment in terms of yield, grain weight,
manure. The seaweed components are micro- and macro- and fresh weight compared to that in the control group. On
nutrients, amino acids, vitamins, auxins, cytokinin, absci- the other hand, applying 100% v/v microalgal liquid extract
sic acid (ABA), and other substances which play a crucial was the best treatment in terms of spike weight, dry weight,
role in plant growth and crop yield [27, 28]. These extracts and grain weight compared to the control [31, 49, 50].
have increased crop yield, seed germination, resistance to Oliveira et al. [32] studied the production of red beet
fungal disease, and insect attack [29]. The seaweed liquid (Beta vulgaris L.), in an organic farming system, assess-
fertilizer (SLF) was prepared according to the published ing biostimulant containing the cell extract of microalgae

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Biomass Conversion and Biorefinery

Fig. 9  Growth analysis during flowering stage in foliar spray method of application

Arthrospira spp. The treatments consisted of two concen- and root growth when compared to those in the control
trations (1.5 and 3.0 g ­L−1) by foliar spray application of group, whereas the Arthrospira species liquid extract treat-
biostimulants and without any treatment, a control group. ment shows a higher number of flowers and the root dry mat-
Treatments were carried out every 2 weeks and evaluating ter. These results suggested that the foliar spray method of
the following traits: dry and fresh weights and the diameter application of S. almeriensis extract can increase the bloom-
of hypocotyls. The application of biostimulant containing ing rate of petunia [33].
the microalgae Arthrospira species elicits higher fresh and Acutodesmus dimorphus microalgal extract was applied at
dry weights when compared to that in the untreated control a concentration of 3.75 g ­L−1 by foliar spray method, dem-
group [32]. onstrating increased tomato plant growth in terms of plant
In ornamental plants, foliar spray treatment using Scened- height and branches, and also a higher number of flowers
esmus almeriensis and Arthrospira species liquid extracts [34]. A. dimorphus extract acts as a seed primer, by trigger-
at the dose of 10 g ­L−1 was evaluated for the production ing seed germination faster, 2 days earlier than the untreated
of petunia. The method of foliar sprayings was done at dif- control [34].
ferent time points such as 0, 14, 28, 35, and 42 days after Furthermore, Arthrospira fusiformis extract was tried
transplanting the plants. The results of S. almeriensis extract on field experiments to evaluate the shelf-life of garlic
showed earliness of flowering, and increased rates of shoot plants and yield. Biostimulants were applied as foliar spray

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 Biomass Conversion and Biorefinery

Fig. 10  Yield analysis

after 1 month of planting and repeated three times at every according to their results [35]. Earlier studies on auxin and
15-day intervals throughout the period of growth. The foliar gibberellic acid from seagrass demonstrate the effect of
spray methods of application tend to increase plant height seedlings in P. australis [52].

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Biomass Conversion and Biorefinery

Fig. 11  Yield analysis in foliar spray method of application

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 Biomass Conversion and Biorefinery

Fig. 12  Chlorophyll estimation in Abelmoschus esculentus plant

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Biomass Conversion and Biorefinery

5 Conclusion Data availability  The datasets generated during and/or analyzed dur-
ing the current study are available from the corresponding author on
reasonable request.
Seagrass consist of all six phytohormones (SA, GA, ABA,
ZE, IAA, and JA) and also micro- and macronutrients in
Declarations 
it. Seagrass showed best in terms of yield (length of pod,
weight of pod, number of flowers and pods) as compared Conflict of interest  The authors declare no competing interests.
to Sargassum, Turbinaria ornata, and Halimeda microloba.
Foliar spray treatment of seagrass was shown to increase the
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