2. REVIEW OF LITERATURE

A review of previous works presented here is by design

restricted to recent studies on Ulva reticulata, Hypnea musciformis and

Sargassum wightii with special reference to physico-chemical parameters

of seawater, mineral distribution in Seaweed liquid fertilizer, Biochemical,

Effect of seaweed liquid fertilizer in experimental studies, and

Phycosynthesis of Silver Nanoparticles, for a complete review is impossible.

The oceans provide unlimited space for capturing solar

energy by marine plants through photosynthesis. Marine plants comprise

of algae, sea grasses, mangroves and sand dune vegetation. The algae

are different shapes and sizes. The microscopic algae, is known as

phytoplankton and macroscopic ones as seaweeds. Most people come

in contact with seaweeds that are washed ashore by the incoming

tides (John Peter Paul and Patric Raja, 2012).

Seaweeds are marine macro algae and primitive type of

plants, growing abundantly in the shallow waters of sea, estuaries and

backwaters. They flourish wherever rocky, coral or suitable substrata

are available for their attachment. Seaweeds are one of the most important

and abundantly available marine living resources. They are not fully

exploited and underutilized (Kaliaperumal et al., 2004). Seaweeds are

classified into three groups’ viz. green algae, brown algae and red algae

and each of these groups differs with regard to their reserve food material

and cell-wall polysaccharides (Jimenez-Escrig and Sanchez-Muniz, 2000).

Seaweeds grow in the intertidal as well as in the subtidal

area up to a certain depth where 0.1% photosynthetic light is

available. Seaweeds are the first organism in the marine food chain, which

provide nutrients and energy for other living organisms (Cheong-xin

chan et al., 2006). They are one of the ecologically and economically

important living resources of the world's oceans. Being the oldest

family of plants on earth, they have admirable qualities of being flexible,

tenacious and prolific (John Peter Paul and Patric Raja, 2012).

Seaweeds provide shelter and habitat for many coastal

animals. Seaweeds have many direct uses, traditionally consumed in

different parts of the world. Recently human consumption of green

algae (5%), brown algae (66.5%) and red algae (33%) is higher in Asia,

mainly in Japan, China and Korea. In Asian countries, seaweeds are

often consumed as marine vegetables (Marinho-Soriano et al., 2006).

Seaweeds are major coastal resources which have been

utilized for the extraction of phycocolloids as alginates from brown algae,

agar and carrageenan from red algae. The major stress on seaweeds in

the coastal zone of Tamil Nadu is pollution, through various means.

90 species of seaweeds belonging to chlorophyceae, phaeophyceae and

rhodophyceae were identified from Hare Island of Tuticorin (Sheela

and Punitha, 2013). Sreekaladevi et al., (2004) also studied the

distribution of marine macroalgae in Idinthakarai and Vizhinjam coasts.

Seasonal variation in zonation, vertical distribution and

biomass of the seaweeds of the Tiruchendur coast was studied by

Krishnamurthy and Balasundaram (1990). Balakrishnan et al., (1992)

studied the distribution and standing crop of algae from six stations

for a period of six months at Muthupet estuary, Tamil Nadu from

March to August, 1988 and recorded 19 species belonging to the

groups of Chlorophyta, Phaeophyta, Rhodophyta and Cyanophyta.

2.1. PHYSICO-CHEMICAL PARAMETERS OF SEAWATER

Seaweeds occur attached to the sea-bed principally in the

intertidal zones where adequate light can penetrate the water column

for supporting their growth. Among the environmental factors light,

temperature, salinity, water motion and nutrient availability are the

major factors affecting their growth in the natural habitats. Seaweeds

grow in diverse light regimes. The water quality and flood of tides have

profound effects on the quantity and quality of light that reaches the

seaweeds (Larkum and Barrett (1983)).

Salinity in the open sea surface is generally 30-34 ppt but

certain seas have markedly higher or lower salinities, because of

evaporation and freshwater influx which regulate the type of seaweed

to occur. Many seaweeds show changes in the metabolic and ionic

concentration in response to the salinity changes of seawater (Reed

and Collins, 1980).

The monthly surface water, temperature fluctuations were

different from the west coast of India as compared to the east coast of

India (Jayasankar and Kulandaivelu, 1999). The nutrient contents vary

with species, geographical location, season and temperature (Dawes et al.,

1993; Kaehler and Kennish, 1996; Haroon, 2000).

Generally, bioaccumulation of the elements in the seaweeds

depends upon the pH, salinity, dissolved oxygen and osmotic potential

of the system (Mabeau and Fleurence, 1993). Foster (1976) investigated

the differences in element concentration of seaweeds in the study

area, during the various seasons might be related not only to different

mineral level in water, but also to different ecological conditions such

as tidal range, temperature and salinity.

2.2. Analysis of Minerals in Seaweed Liquid Fertilizer (SLF)

Seaweeds are important marine renewable resources. They

are used as food, feed, fodder, fertilizer, agar, alginate, carageenan and

source of various fine chemical (Sahoo, 2000). In recent years, the use

of natural fertilizer has allowed for substitution in place of conventional

synthetic fertilizer (Hong et al., 2007). Seaweed extracts are marketed

as liquid fertilizers and biostimulants since they contain many growth

regulators such as cytokinins (Durand et al., 2003; Strik et al., 2004).

Long term use of synthetic chemical fertilizers will damage

the physico-chemical character, microflora and their micro-ecology of

soil. Chemical fertilizers are capable of killing off many of the soil

organisms that are responsible for decomposition and soil formation.

Naturally occurring organic fertilizers differ from chemical fertilizers, in

that; they feed the plants while building the soil’s structure

(Kamaladhasan and Subramanian, 2009).

Seaweeds contain good amount of nitrogen, potassium and

other minerals and trace elements, and also the carbohydrates and

other organic matters present in seaweeds helps in altering the nature

of soil and improving its moisture retaining capacity (Simpson and

Hayes, 1958). Seaweed extracts are known to enhance seed germination

and growth, increased uptake of nutrients, impart a degree of frost

resistance and make plants to withstand better towards Phytopathological

fungi and insect pests (Bhosle et al., 1975). Apart from macro and

micro nutrients seaweeds contain many growth promoting hormones

like cytokinin, gibberellins, auxins (Tay et al., 1987).

Seaweed liquid fertilizer is a bio-organic mixture that contains

many growth promoting substances like auxins, gibberellins, trace

elements, vitamins and amino acids (Metting et al., 1990; Selvaraj et al.,

2004; Sylvia and Baluswami, 2005). The seaweed extracts contain plant

growth hormones, regulators, promoters, carbohydrates, amino acids,

antibiotics, auxins, gibberellins and vitamins consequently enhance the

yield and quality which induce the yield of crops, seed germination,

resistance to frost, fungal and insect attacks (Erulan et al., 2009).

Seaweeds are commercially economic important, renewable marine

resource. Variability in chemical constituents and growth of algae may

be interspecific, intra-annual or inter-annual. Certain seaweeds contain

significant quantities of proteins, lipids, minerals and vitamins

(Norziah and Ching 2002; Sanchez- Machado et al., 2004; Van

Ginneken et al., 2011).

The liquid extract of brown alga Hydroclathrus clathrus

increased chlorophyll a and b contents of the leaves of Sorghum

vulgare (Ashok et al., 2004). Iron, copper and magnesium are the essential

elements which act as catalyst for the synthesis and maintenance of

chlorophyll (Paul and Nangkynrih, 1996). Nickel at low level also

increases the chlorophyll (Narwal et al., 1996).

Plant nutrition is one of the most important factors that

increase plant production. Nitrogen plays the most recognized roles in

the plant for its presence in the structure of the protein molecule. In

addition, nitrogen is found in such important molecules as purines,

pyrimidines, porphyrines and coenzymes. The porphyrin structure is

found in metabolically important compounds such as chlorophyll

pigments and the cytochromes which are essential in photosynthesis

and respiration (Marschner, 1995). Nitrogen availability is often a limiting

factor in crop productivity, particularly in developing countries where

nitrogen fertilizers are either unavailable or unaffordable (Graham,

1981). Nitrogen fertilization also plays a significant role in crop quality.

The recommendations in both tropical and temperate areas are that

farmers would apply to crops at least minimal doses of nitrogen

(Vance, 1998; Fink et al., 1999). It has found wide application in modern

agriculture for the use of marine macroalgae as fertilizer. Seaweed

contains all the trace elements and plant growth hormones required

by plant, regulators promoters available to enhance yield attributes

(Crouch and Van Staden, 1991 and 1993).

Seaweed requires inorganic carbon, water, light and various

mineral ions for photosynthesis and growth. Nitrogen, Phosphorus,

Potassium, Calcium, Magnesium, Sulphur, Iron, Manganese, Copper,

Zinc, Molybdenum, Sodium, Chlorine, Boron, Cobalt found in the sea

are suitable for growth of seaweeds. Some seaweed requires trace

amounts of one or two organic carbon compounds or vitamins for

their growth. Vitamin B12 is also required by seaweeds for their growth

which is present in the seawater in a trace (Lobban and Harrison,

1994). Bio-fertilizers enhance crop productivity through processes

such as nitrogen fixation, phosphate solubilisation and plant hormone

production (Pereira and Verlecar 2005).

2.3. Preparation, mode of application and growth promoting role of SLF

Seaweed can be added in the form of fresh and dried or

burnt material to the soil as fertilizer for growing peanuts and sweet

potatoes (Tseng, 1973). Use of seaweeds as manure is a common

practice in coastal areas of Tamil Nadu and Kerala for coconut

plantations. The method of preparation and properties of seaweed liquid

fertilizers from Sargassum was given by Sreenivasa Rao et al., (1979).

Caisozzi et al., (1980) used the marine algae Macrocystis pyritera as

fertilizer on corn and studied the growth parameters. Inorganic fertilizers

and combination of seaweed manure in Pennisetum typhoides and

Arachis hypogaea found that the performance of treatments was better

than other treatments (Bokil et al., 1972).

Seaweed extracts exhibit growth stimulating property of

crop plants. Hence its formulation can be used as a bio-stimulant in

agriculture. Bio-stimulant is defined as a ‘material’ other than fertilizer

that promotes the growth and yield attribute property of the plants

when applied in a small quantity during a crop cycle. The biostimulant

present in seaweed extract increase the vegetative growth (10%), the

leaf chlorophyll content (11%), the stomata density (6.5%), photosynthetic

rate and the fruit production (27%) of the plant (Spinelli et al., 2010).

In spite of the proven capability of the SLF on growth and

yield promotion of various crops, the extraction procedure from

seaweeds, its concentration and mode of application have not been

standardized. The liquid seaweed extracts from seaweeds are usually

prepared by hydrolyzing the material under pressure. However, the

preparation may vary from species to species depending upon the

amount of dried material available. Its method of extraction significantly

differs from person to person and also the mode of application to

crops. Seaweed extracts are used in several ways, such as drench in soil

during transplantation, during field preparation (Lingakumar et al., 2002).

The growth promoting effect of the liquid extracts of

seaweeds on seed germination (Mostafa et al., 1999). Efforts have been

made to enhance growth and yield of tomato plants and to improve

lycopene and vitamin C content of fruits, by treatments with aqueous

extract of Sargassum johnstonii (Kumari et al., 2011). The diluted liquid

seaweed extract also enhanced the plant’s defense against diseases

and increases salt (Jayaraman et al., 2011) and drought tolerance (Kumar

and Mohan, 2000). An adequate amount of growth promoting hormones

and micronutrients present in seaweed makes them an excellent fertilizer.

Unlike chemical fertilizer, seaweed extract are biodegradable, non-toxic,

non-polluting and non-hazardous to human (Dhargalkar and Pereia,

2005).

Foliar spray application of mineral nutrients offers a

quicker method of supplying nutrients to higher plants than methods

involving root application. The preferred mode of foliar absorption of

nutrient elements is still under debate. Recently, some authors

pointed out the possibility of an active uptake through stomatal pores

instead of cuticular uptake (Eichert et al., 1998 and Burkhardt and

Eichert, 2001).

Aqueous extract of Sargassum wightii when applied as a

foliar spray on Zizyphus mauritiana showed an increased yield and

quality of fruits (Rama Rao, 1991). Growth promoting effect of seaweed

liquid fertilizer Enteromorpha intestinalis on the sesame crop plant

(Gandhiyappan and Perumal, 2001). Seaweed foliar applications

increased harvestable bean yields by an average of 25% (Temple and

Bomke, 1989). Kannan and Tamilselvan, (1990) observed that the soil

application of SLF of Enteromorpha clathrata and Hypnea musciformis

increased the growth characteristics of green gram, black gram and rice.

Fornes et al., (2002) stated the beneficial effect of seaweed

extract application as a result of many components that may work

synergistically at different concentrations, although the mode of action

still remains unknown. In recent years, uses of seaweed extracts have

gained in popularity due to their potential use in organic and sustainable

agriculture (Russo and Beryln, 1990). In an experiment with cluster

beans, it was found that by the use of 1.5% Sargassum wightii and

1.0% of Ulva lactuca growth was increased, but the highest

concentrations retarded the plant growth due to stress and wilting of

leaves (Sivasankari Ramya et al., 2010). When dealing with the effect

of SLF on seed germinations in crops, red seaweed, green seaweed and

brown seaweed also yielded slightly different results due to differences

in the chemical constituent between the species (Demir et al., 2006).

Seaweed extracts are bioactive at low concentrations

(diluted as 1:1000 or more) (Crouch and van Staden 1993). Although

many of the various chemical components of seaweed extracts and their

modes of action remain unknown, it is plausible that these components

exhibit synergistic activity (Vernieri et al., 2005). SLF treatment

increased the number of branches and concentration of photosynthetic

pigments (Sridhar and Rengasamy, 2010). Galbiattia et al., (2007)

focussed crop cultivation using organic fertilizers has contributed for

deposition of residues, improving physical and chemical properties of

soil that is important for biological development.

Seaweeds and seaweed products enhance plant chlorophyll

content (Blunden et al., 1997). Application of a low concentration of

Ascophyllum nodosum extract to soil or on foliage of tomatoes produced

leaves with higher chlorophyll content than those of untreated controls.

This increase in chlorophyll content was a result of reduction in

chlorophyll degradation, which might be caused in part by betaines in

the seaweed extract (Whapham et al., 1993). Glycine betaine delays

the loss of photosynthetic activity by inhibiting chlorophyll degradation

during storage conditions in isolated chloroplasts (Genard et al., 1991).

Although they may contain different levels of minerals, biostimulants

are unable to provide all the nutrients needed by a plant in required

quantities (Schmidt et al., 2003); however, their main benefit is to

improve plant mineral uptake by the roots (Vernieri et al., 2005) and

in the leaves (Mancuso et al., 2006).

In recent years, liquid extracts prepared from different

seaweeds have started gaining importance as foliar sprayers or soil

conditioners for several important crops (Rama Rao 1991, Mohan et al.,

1994, Rajkumar and Subramanian 1999, Thirumaran et al., 2006,

2007, 2009, Rathore et al., 2009, El-Quesni et al., 2010). The use of

seaweed extract at the germinating stage showed encouraging results

by stimulating the growth of roots and shoots (Featonby and Van

Staden, 1983). In some developing countries, about 2-10% of seaweed

extracts enhance the yield of the crop of the commercially important

plants (Chatterji et al., 2004).

Seaweed concentrates triggers early flowering and fruit set

in a number of crop plants (Abetz and Young 1983; Featonby-Smith

and van Staden 1987; Arthur et al., 2003). Fertilizers differ from plant

growth regulators in several ways, the growth regulators involves in

cell division, root and shoot elongation, initiation of flowering and

other metabolic function, the fertilizers simply supply the minerals

needed for normal growth of the plant. Therefore, seaweed liquid

fertilizers, a blend of both plant growth regulators and organic nutrient

input is eco-friendly promoting sustainable productivity and maintaining

the soil health. The extracts of Sargassum sp., Sargassum polycystum,

Hydroclathrus sp., Turbinaria ornata and Turbinaria murrayana, were

able to induce the growth of rice plants (Sunarpi et al., 2010).

Liquid seaweed extract when applied to seed, soil or

sprayed on crops, increased seed germination percentage, nutrient

uptake, growth (Rajkumar Immanuel and Subramanian, 1999) and

yield of crops (Anantharaj and Venkatesalu, 2002). Seaweed liquid

fertilizer exhibits growth stimulating property on crop plants. Hence

its formation can be used as a bio-stimulant in agriculture. Bio-

stimulant is defined as a ‘material’ other than fertilizer that promotes

the growth and yield attribute property of the plants when applied in a

small quantity during a crop cycle. The bio-stimulant present in seaweed

liquid fertilizers increase the vegetative growth, leaf chlorophyll

content and fruit production of the plant (Spinelli et al., 2010).

Abdel-Mawgoud et al., (2010) investigated Seaweed extract

can be used as a growth enhancer for a variety of plants at a lower

concentration without any harmful effects. Plants sprayed with

seaweed extract showed healthy growth with bright green and larger

leaves, early flowering and fruit bearing as compared to the group

where no seaweed extract was used. The use of natural seaweed products

as substitutes of the conventional synthetic fertilizers has assumed

importance. In agriculture, the application of seaweeds are so many,

as soil conditioners, fertilizers and green manure, due to the presence

of high amount of potassium salts, micronutrients and growth

substances. Seaweed liquid fertilizer contained macronutrients, trace

elements, organic substances like amino acids and plant growth

regulators such as auxin, cytokinin and gibberellins. They are

particularly suitable content (Chapman and Chapman, 1980); it has

been proved that SLF promoted the growth and the yield of crop

plants (Rama Rao, 1991; Rama Rao, 1992).

Cytokinins in vegetative plant organs are associated with

nutrient partitioning, whereas in reproductive organs, high levels of

cytokinins may be linked with nutrient mobilization. Fruit ripening

generally causes an increase in transport of nutrient resources within

the developing plant and the fruits have the capacity to serve as

strong sinks for nutrients (Adams-Phillips et al., 2004). Photosynthate

distribution could be shifted, perhaps markedly, moving from

vegetative parts (roots, stem, and young leaves) to the developing fruit,

to be utilized in fruit development (Nooden and Leopold, 1978).

Seaweeds are rich in cytokinin and increase its availability to plants

eventually results in a greater supply to the maturing fruit. Developing

fruits and seeds demonstrated increased endogenous cytokinin levels

(Letham, 1994). It has been reported that the increased cytokine

concentration is associated with translocation of cytokinin from the

roots to other plant parts (Carlson et al., 1987).

Seaweed manure has the advantage of being free from

weeds and pathogenic fungi. Liquid extracts of brown algae are being

sold as biostimulants or biofertilizers in various brand names.

Promising increased crop yield, nutrient uptake, resistance to frost

and stress, improved seed germination of reduced incidents of fungal

and insect attack have been resulted from application of seaweed

extracts. Seaweeds are known to contain appreciable quantities of

plant growth regulators (Mooney and Van Staden, 1985).

Recent research suggests that application of seaweed

extract as seed treatment and/or foliar spray helps significant growth

of plants. The extract contains micro-nutrients, auxins and cytokinins

and other growth promoting substances (Spinelli et al., 2010). These

hormones play an important role in enhancing of cell size and cell

division, and together they complement each other as cytokinins are

effective in shoot generation and auxins in root development, while

micro-nutrients improve soil health (Liu and Lijun, 2011). Sheela and

Punitha, (2013), the use of biofertilizer will be helpful to sustain soil

fertility and the pollution free soil environment. Seaweed extracts from

Sargassum wightii and Ulva fasciata, have been found to increase the

yield of C3 plant Phaseolus mungo. The different growth promoters

Auxin, gibberellins have been reported and enhance growth of C3

plants.

Seaweed fertilizers are one of the natural organic fertilizers

containing highly effective nutritious and promotes faster germination

of seeds, increase yields and resistant ability of many crops.

Biofertilizers enhance crop productivity through processes such as

nitrogen fixation, phosphate solubilisation and plant hormone

production (Pereira and Verlecar, 2005). Liquid fertilizers derived from

seaweeds are found to be superior over chemical fertilizer due to high

level of organic matter, macro and micro elements, vitamins, fatty

acids and also the growth regulators (Barkett and Vanstaden, 1990;

Maria Victorial Rani and Revathy, 2009). Seaweed application would

increase the trace element content in the crop plants (Johnsi

Christobel, 2008).

Application of Maxicrop enhanced harvestable yield in

lettuce, whereas an increase in the heart size of the florets and curd

diameter was observed in cauliflower (Abetz and Young, 1983).

Similarly, a substantial increase in yield was achieved in barley

(Featonby-Smith and Van Staden, 1987) and peppers (Arthur et al.,

2003) after treatment with Kelpak. Foliar application of seaweed liquid

extract (Kelpak 66) enhanced bean yield by 24% (Nelson and van

Staden, 1984). Kelpak 66 also had a similar effect on the yield of

wheat under potassium stress, although its application had no

significant effect on the plants receiving an adequate K supplement

(Beckett and van Staden, 1989).

Seaweed extracts are now available commercially under the

names, such as Maxicrop (Sea born), Algifert (marinure), Goemar

GA14, Kelpak 66, Seaspray, Seasol, SM3, Cytex and Seacrop 16.

Recently, researchers have proven that seaweed fertilizers are better

than other fertilizers and are very economical (Gandhiyappan and

Perumal, 2001). Seaweed liquid fertilizers are enhancing the growth

and yield of certain commercial crops (Sridhar and Rengasamy, 2010;

Sangeetha and Thevanathan, 2010).

As India is an agricultural country, nearly 70% of the

population thrives in rural areas engaged in agriculture making the

backbone of our economy. The fast growing population is mounting

tremendous pressure on food production in the country. To meet out

this increasing demand, farmers use chemical fertilizers to enhance

the crop production. In agriculture, the application of seaweeds is

used as fertilizers and green manure due to the presence of high

amount of minerals, micronutrients and growth substances. The

growing agricultural practices need more fertilizers for higher yield to

satisfy food for human beings. Hence marine algae, particularly

seaweeds have a vital role to play in agriculture, especially in the third

world country where the irrational use of chemical fertilizer and

pesticides is a cause of concern. Extensive regional tribals would need

to be conducted with the product to determine the environmental

limits on biological activity and monitor the survival and dispersal of

the inocula (Davison, 1988). Hence the use of modern agriculture in

conjunction with traditional farming practices is the sustainable

solution for the future. Application of seaweed extract as organic

biostimulant is fast becoming accepted practice of horticulture due to

its beneficial effects (Verkleij, 1992).

An effect of seaweed extracts on morphology of plants such

as germination of seeds, root and leaves of seedlings. Jayachandran

and Ramasamy (1999) investigated the effects of extract of Hypnea

musciformis on the epidermal morphology and frequency of root

nodules in addition to its effects on other morphological characters of

seedling in Arachis hypogea.

Stomata are the minute units of the epidermal tissue

system. These are the openings in the epidermis, limited by the two

specialized cells termed the guard cells. The guard cells together with

the opening form stomata. The guard cells have uneven thickened

walls. The guard cells also covered with cuticle which extends to the

inner wall forming the boundary of the pore and sub stomatal

chamber. Stomata consist of a pore surrounded by two guard cells.

The epidermal cells adjoining the guard cells are called the subsidiary

cells. The stomata together with the subsidiary cells termed as stomatal

complex. Below the stomata and directed inwards to the mesophyll are

larger intercellular spaces which are termed as sub stomatal chambers.

On the basis of number and arrangement of the subsidiary cells, Metcalfe

and Chalk, (1950) found the different types of stomata viz. Anomocytic,

Anisocytic, Diacytic, Paracytic, Actinocytic and Cyclocytic.

Farmers, all throughout the world are switching over to

organic fertilizers. Seaweed manure besides increasing the soil fertility

increases the moisture holding capacity and supplies adequate trace

metals thereby improving the soil structure. This explains its worldwide

use as manure along the coastal areas. Recently adopted technique, of

spraying fertilizer on the plants has increased nutrient absorption

efficiency in the plants. The nutrients are not leached down into the

soil, but are available to the plant through leaf openings such as lenticels,

hydathods and stomata. Leaves absorb nutrients within 10 to 15

minutes of its application (Ganapathy selvam and Sivakumar, 2013).

Black gram belongs to family Leguminoseae and subfamily

Papilionaceae. It is an annual herb growing to a height of 30-100 cm.

The stem is slightly ridged and covered with brown hairs, leaves are

large and trifoliate. The inflorescence consists of a cluster of 5-6 flowers at

the top of a long hairy peduncle. The flower of black gram is bright

yellow and the pod attaches upright to the peduncle. Black gram is a

self-fertilized crop. Black gram may be grown as pure crop in rice fallows

after the harvest of the first or second crop of paddy. It can also be

grown as a pure or mixed crop during kharif season. The area of

traditional cultivation of black gram is confined to South Asia and

adjacent regions (India, Pakistan, Afghanistan, Bangladesh and Myanmar

(Rajarathinam and Rathnaswamy, 1999).

Seed pelleting commonly applicable technique in direct

sown crops, also provides an opportunity to package effective quantities of

material (inputs) which supplies not only micro and macro nutrients

but also protects the crop from pests and disease during the early stages.

The density and operation (opening) of stomatal pores on leaf surfaces

are both heavily influenced by environmental cues. Together, they control

the leaf stomatal conductance to water vapour over short (minute to

hour) and long (seasonal to lifetime) timescales (Casson and Hetherington,

2010) and enable the plant to balance the conflicting needs to capture

atmospheric carbon dioxide for photosynthesis and to minimize water

loss through transpiration. Plants maintain plasticity in their capacity

to moderate stomatal density during leaf growth and, although stomatal

density correlates with the macro-environment over geological timescales

(Hetherington and Woodward, 2003), there is also a strong inverse

correlation with water use efficiency (WUE) during growth and

development (Miyazawa et al., 2006; Lake and Woodward, 2008). The

frequency of stomata on the leaf epidermis (Stomatal Index, SI: stomata

as a percentage of epidermal cells) responds to light, CO2 concentration,

drought, and evaporative demand - relative – humidity (Royer, 2001;

Casson et al., 2009).

Scanning Electron Microscopic studies on some species of

Hypnea Lamouroux were made by Prema et al., (2000). The surface

features of the thallus at the apical end of the ramuli of Hypnea

valentiae featured the outer thick cuticle. The uneven surface of the

peripheral fractured thallus showed the single layer and some areas

showed double layered cortical cells. The dark nature of these cells

represents the presence of chromatophores. Selvaraj et al., (2007)

studied Electron Microscopic studies and X-ray microanalysis of

Stoechospermum marginatum and Gracilaria corticata with reference to

cell structure and cell wall organization and composition.

Energy dispersive spectroscopic analysis (EDS) provides a

unique approach for obtaining quantitative compositional analysis of

individual cell and intracellular compartments. Elemental quantification

of semi-thin sections with electron probe X-ray microanalysis (EPMA)

in generally based on the linear relationship between elemental

concentration and the ratio of the number of characters/continues

like X-ray photons (shuman et al., 1976; Silverberg, 1976; Kitazawa et al.,

1983). The macro and micro elements are generally regarded as being

cell wall associated and has been detected by XRMA in a range of algal

cells, including blue, green algae (EI-Bestway et al., 1996; Krivtso et al.,

2000). The amount of different chemical elements present in the leaf

of the Vigna mungo was determined using EDS in the present study.

2.4. ANALYSIS OF PHYCOSYNTHESIS OF SILVER NANOPARTICLES

The field of nanotechnology is an immensely developing

field as a result of its wide-ranging applications in different areas of

science and technology. The word, nanoparticle (10-9m) can be defined

in nanotechnology as a small object that acts as a whole unit in terms

of its transport and properties. The word “nano” is derived from a

Greek word meaning dwarf or extremely small (Rai et al., 2008).

Nanotechnology has a wide variety of applications in various

fields like optics, electronics, catalysis, bio-medicine, magnetics,

mechanics, energy science, etc. Nanobiotechnology is a multidisciplinary

field involving research and development of technology in different

fields of science like biotechnology, nanotechnology, physics, chemistry,

and material science (Huang et al., 2007; Jain et al. 2011). It deals

with bio-fabrication of nano-objects or bi-functional macro-molecules

used as tools to construct or manipulate nano-objects. Since, microbial

cells offer many advantages like wide physiological diversity, small

size, genetic manipulability and controlled culturability; they are thus

regarded as ideal producers for the synthesis of diversity of nanostructures,

materials and instruments for Nanosciences (Villaverde, 2010).

The green synthesis of AgNPs involves three main steps,

which must be evaluated based on green chemistry perspectives,

including (1) selection of solvent medium, (2) selection of environmentally

benign reducing agent, and (3) selection of nontoxic substances for the

AgNPs stability (Raveendran and Wallen, 2003; Li et al., 2007). Sinha

et al., (2009) reported the importance of bio-inspired synthesis is

being emphasized at present and made to design a protocol for “green

synthesis” in which there is no involvement of high pressure,

temperature and toxic chemicals.

Nanotechnology is currently employed as a tool to explore

the darkest avenues of medical sciences in several ways like imaging,

sensing, targeted drug delivery, gene delivery systems and artificial

implants. By manipulating materials at the atomic level, nanotechnology

offers to achieve unique properties for various desired applications in

biology (Gleiter, 2000). Nanotechnology is now poised to enter a

commercialization era. NPs are showing promise in different fields of

agricultural biotechnology (Rahman et al., 2009). Most of the nature’s

creation occurs at the nanoscale regime, thus fusion between

nanotechnology and biology can mimic nature and bring about a

revolution in the field of health and medicine, for example, in drug

delivery (Dhar et al., 2008), cancer therapy (Mukherjee et al., 2005)

and medical diagnostic kits (Roe et al., 2008). The inorganic antibacterial

agents have the advantage over the organic antimicrobial agents in

terms of their stability, toxicity, preparation methods, and so on

(Anagnostakos et al., 2008).

Nanoparticles are being viewed as fundamental building

blocks of nanotechnology. The most important and distinct property of

nanoparticles is that they exhibit larger surface to volume ratio. Silver

has long been recognized as having an inhibitory effect toward many

bacterial strains and microorganisms commonly present in medical

and industrial processes (Mostafa et al., 2011). The most widely used

and known applications of silver and silver nanoparticles include

topical ointments and creams containing silver to prevent infection of

burns and wound (Murphy, 2008).

Production of nanoparticles can be achieved through

different methods, for example reduction in solutions, chemical and

photochemical reactions in reverse micelles, thermal decomposition of

silver compounds (Plante et al., 2010), radiation-assisted (Cheng et al.,

2011), electrochemical (Hirsch et al., 2005) and recently via green

chemistry methods (Sivakumar et al., 2012).

Nanoparticles are classified primarily into two types, viz

organic and inorganic nanoparticles. The nanoparticles of carbon are

called the organic nanoparticles. Magnetic nanoparticles, noble metal

nanoparticles (platinum, gold and silver) and semiconductor nanoparticles

(titanium dioxide, zinc oxide and zinc sulfide) are classified as

inorganic nanoparticles (Kathiresan and Asmathunisha, 2013). Biological

synthesis is cost-effective, environmental friendly and easily scaled up

for large-scale synthesis. In this method there is no need to use high

pressure, energy, temperature and toxic chemical that may have adverse

effect in medical applications. Particularly, silver (Ag) nanoparticles are

outstanding with unique optical, electrical, thermal and electromagnetic

properties (Pradeep and Anshup, 2008; Choi et al., 2007; Reddy et al.,

2008). The metal nanoparticles have magnetic, electronic and optical

properties, which make their usage in different fields like medicine,

agriculture and electronics (Rai et al., 2012). Among the metal

nanoparticles, silver nanoparticles have gained remarkable consideration

owing to their physicochemical properties (Elechiguerra et al. 2005).

Nanotechnology involves the creation and manipulation of

materials at nanoscale levels to create products that exhibit novel

properties. Recently, nanomaterials such as nanotubes, nanowires,

fullerene derivatives (buckyballs) and quantum dots have received

enormous attention to create new types of analytical tools for

biotechnology and life sciences (Bruchez et al., 1998; Taton et al.,

2000; Cui and Lieber, 2001).

Nanomaterials are currently being widely used in modern

technology; there is a serious lack of information concerning the

human health and environmental implications of manufactured

nanomaterials. Nanotechnology is the manipulation, integration or self-

assembly of individual atoms, molecules, or molecular clusters into

complex to create material with new and extremely diverse properties.

“Nano” suffix usually refers to a size scale between 1-100 nm in

dimension (Tarafdar and Raliya, 2012).

Silver is widely known as a catalyst for the oxidation of

methanol to formaldehyde and ethylene to ethylene oxide (Nagy et al.,

1999). Nanoparticles of silver are widely used in water filters (Jain and

Pradeep, 2005), bio sensors (Chen et al., 2007), antibacterial activity

(Venkatpurwar and Pokharkar, 2011), anti-HIV activity (Elechiguerra

et al., 2005) and in controlling plant pathogens (Krishnaraj et al.,

2012). Nanoparticles could also be stabilized directly in the process by

proteins (Duran et al., 2005). Biomolecules as reducing agents are

found to have a significant advantage over their counterparts as

protecting agents. At this size and dimensional range, essentially any

material will exhibit different properties from those it would as an

atomic cluster or as the larger bulk materials (Rajesh et al., 2012).

In the past few decades, several amount of antibiotics used

for the treatment of human diseases; resulted to create many pathogenic

bacteria resistant to multiple drugs. Now-a-days, the multidrug

resistance bacteria are developed due to the bacterial transposons

occur on resistance (R) plasmids and also over expression of gene that

code for multidrug efflux pumps. In recent years, multidrug resistant

bacteria are increasingly held responsible for wound infection and

have become a serious public health issue, which raised the need to

develop new bactericidal materials (Nikaido, 2009). Many researchers

have clearly noted that these bacteria capable of communicating

within themselves with the help of quorum sensing molecule and

establishes their infection rapidly in human for its survivability. Most

of gram-positive and gram-negative bacteria capable of producing

small molecules called autoinducers to communicate each other on

when a threshold number of same bacterial species are present. This

molecule, directly or indirectly induce the virulence factors of bacteria,

thereby make the bacteria to survive any environmental stress

(Vattem et al., 2008).

Materials with a particle size less than 100 nm in at least

one dimension is generally classified as nanomaterials. The

development of nanotechnology in conjunction with biotechnology has

significantly expanded the application domain of nanomaterials in

various fields. A variety of carbon-based, metal and metal oxide based

dendrimers (nano-sized polymers) and biocomposites nanomaterials

EPA (Environment Protection Agency), 2007; Nair et al., 2010) are

being developed. Types include single-walled and multi-walled carbon

nanotubes (SWCNT/MWCNT), magnetized iron (Fe) nanoparticles,

aluminum (Al), copper (Cu), gold (Au), silver (Ag), silica (Si), zinc (Zn)

nanoparticles and zinc oxide (ZnO), titanium dioxide (TiO2), and cerium

oxide (Ce2O3), etc. General applications of these materials are found in

water purification, wastewater treatment, environmental remediation,

food processing and packaging, industrial and household purposes,

medicine, and in smart sensor development (Zambrano-Zaragoza et al.,

2011; Bradley et al., 2011). The majority of applications in these areas

have focused on the significance of the nanomaterials for improved

efficiency and productivity. These materials are also used in agriculture

production and crop protection (Bouwmeester et al., 2009; Nair et al.,

2010; Sharon et al., 2010; Emamifar et al., 2010).

In recent years, various researchers have studied the effects

of nanomaterials on plant germination and growth with the goal to

promote its use for agricultural applications. Zheng et al., (2005)

studied the effects of nano and non-nano TiO2 on the growth of

naturally-aged spinach seeds. It was reported that nano-TiO2 treated

seeds produced plants that had 73% more dry weight, three times

higher photosynthetic rates, and 45% increase in chlorophyll ‘a’

formation compared to the control over a germination period of 30

days. A precedent exists for conducting comprehensive literature reviews

as a guide to the further development of nanomaterials applications.

Reviews are available involving water disinfection (Li et al., 2008), the

food industry (Sanguansri and Augustin, 2006), non-point source

pollution control (Shan et al., 2009), treatment of environmental waste

(Macaskie et al., 2010), and the design of trace concentration

detection devices (Zhang and Fang, 2010).

In the past decade, several kinds of the biological organisms

like microbes, plants and seaweeds have been employed and well-

studied for the synthesis of Ag nanoparticles (Ramanathan et al., 2011;

Ahmad et al., 2003; Shankar et al., 2003; Mohanpuria et al., 2008;

Kumar et al., 2012a). Utilization of plants for the synthesis of Ag

nanoparticle is advantageous over other biological methods. The rate of

biosynthesis of Ag nanoparticles from plants is cost effective and does

not use toxic chemicals, temperature and high pressure (Parashar et al.,

2009). Several compounds include primary and secondary metabolites

synthesized by seaweeds are a promising source for both industrial

and biotechnological applications (Renn, 1997).

Seaweeds or benthic marine algae are the group of plants

that live either in marine or brackish water environment. The synthesis of

nanoparticles using algae as source has been unexplored and

underexploited. Singaravelu et al., 2007 reported the extracellular

synthesis of monodisperse gold nanoparticle size of 8-12 nm using

marine algae, Sargassum wightii in short duration and proved that the

nanoparticle synthesized using marine algae found to be more stable

in solution, a very important advantage over other biological methods.

In chemical method, the use of toxic chemicals on the

surface of nanoparticles and non-polar solvents during the synthesis

procedure limits their applications in biomedical and clinical fields.

Therefore, there is a need for the development of clean, safe,

biocompatible, cost effective, nontoxic, sustainable, and environmental

friendly method for synthesizing the nanoparticles. Compared with the

traditional synthetic methods, biological systems provide a novel idea

for the production of nanomaterials (Bansal et al., 2011). Among the

most important bioreductants are plant extracts, which are relatively

easy to handle, readily available, low cost, and have been well explored

for the green synthesis of other nanomaterials (Khan et al., 2013).

Moreover, the biologically active molecules involved in the green

synthesis of NPs act as functionalizing ligands, making these NPs

more suitable for biomedical applications (Lu et al., 2007).

Biologically synthesized silver nanoparticles (Ag-NPs) have

a wide range of applications because of their remarkable physical and

chemical properties. The literature on the extracellular biosynthesis of

Ag-NPs using plants and pure compounds from plants are insignificant

(Kattumuri et al., 2007; Song and Kim, 2008; Gilaki, 2010). The

biological method for the synthesis of nanoparticles employs the use

of biological agents such as bacteria (Beveridge and Murray, 1980),

yeast (Huang et al., 1990), fungi (Frilis and Myers-Keith, 1986) and

alga (Sakaguchi et al., 1979; Darnall et al., 1986) are capable of

absorbing and accumulating metals. The biological agents secrete a

large amount of enzymes, which are capable of hydrolyzing metals and

thus bring about enzymatic reduction of metal ions (Rai et al., 2009).

Bhainsa and D’Souza (2006), reported the among them

silver nanoparticles have wide applications and are employed as

spectrally selective coating for solar energy absorption, optimal receptors

in intercalation material for electrical batteries, polarizing filters,

catalysts in chemical reaction, biolabelling, and as antimicrobial

agents in biomedical field.

In most literature surveys the AgNPs show inhibitory and

bactericidal activity against a wide range of microorganism. Biological

synthesis of silver nanoparticles using bacteria, fungi, algae, enzymes

and plant extracts is ecofriendly, time conception and able to synthesize

different sizes and shapes of stable nanocolloids (Gnanadesigan et al.,

2012). Moreover synthesis of AgNPs using marine brown algae shows

more advantageous over other biological processes because it reduces

the cell maintaining process, easy to harvest and also extremely suitable

for large scale production of silver nanoparticles (Singh et al., 2013).

Dyes belong to the class of synthetic organic compounds

and are widely used in the textile industry. The removal of these non-

biodegradable organic chemicals from the environment is a crucial

ecological problem. Many techniques, such as activated carbon

sorption, flocculation, electro-coagulation, UV-light degradation, and

redox treatments, are being routinely used for abating dyes (Kumar et

al., 2011). However, due to the ineffectiveness of these techniques in

some way or the other, the present scenario requires better and

improved wastewater-treatment measures. Recently, metal nanoparticles

were reported as effective photocatalysts for degrading chemical

complexes, under ambient temperature with visible light illumination

(Mohamed et al., 2012). This can be achieved by increasing the optical

path of photons leading to a higher absorption rate of nanoparticles in

the presence of a local electrical field (Garcia, 2011). Nanoparticles are

metal particles and exhibit different shapes like spherical, triangular,

rod, etc., (Sau and Rogach, 2010). These nanoparticles showed new

and improved properties based on their morphological structures and

characteristics as compared to bulk materials (Gurunathan et al., 2009).

Biological methods for synthesis of nanoparticles and their

role in the biotransformation process of formation of different bio-

products, such as bioethanol, biohydrogen, biodiesel, enzymes and

bioplastics is reported by Mohapatra et al., (2011) because there is an

increasing commercial demand for bio-nanoparticles due to their wide

applicability in various areas and other bio-products. The nanoparticles

are going to prove revolutionary in the field of biotransformation by

improving the efficiency and yield and often widening the application

range. Additionally, the possibility to recover H2 from waste organic

streams in biorefineries using photocatalytic approaches is an attractive

option to enhance process sustainability and produce valuable energy

products. With respect to the overall photoreforming to obtain H2 and

CO2, the photo-dehydrogenation of bioethanol leads to the co-production

of a valuable chemical (acetaldehyde) together with H2 (Ampelli et al.,

2013).

Green chemistry started for the search of benign methods

for the development nanoparticles and searching antibacterial,

antioxidant, and antitumor activity of natural products. Biosynthetic

processes of nanoparticles have received much attention as a viable

alternative for the development of metal nanoparticles where by-

products of factories and plant extract is used for the synthesis of

nanoparticles without any chemical ingredients (Badri Narayanan and

Sakthivel, 2008; Vijayakumar et al., 2013).

Running shoes, socks and even computer keyboard may be

impregnated with silver nanoparticles that can kill some bacteria keep

you swelling sweet and preventing the spread of infection among

computer users. Researchers in India point out those silver nanoparticles

are not only antibacterial against so called Gram Positive bacteria,

such as resistant strains Staphylococcus aureus and Streprococcus

pneumonia, but also against Gram Negative Escherichia coli and

Pseudomonas aeruginosa (Pattabi, 2010). Scientists are reporting

development and successful lab tests of “killer paper” a material intended

for use as a new food packaging material that helps preserve foods by

fighting that bacteria that cause spoilage. The silver coated paper

showed potent antibacterial activity against E.coli and S.aureus, two

causes of bacterial food poisoning killing all of the bacteria in just

three hours (Langmuir, 2011).

The use of nanoparticles derived from noble metals has its

application in many areas, including Jewellery, electronics, medical

fields, water treatment and sport utilities thus improving the longevity

and comfort in human life. The application of nanoparticles as delivery

vehicles for bactericidal agents represents a new paradigm in the design of

antibacterial therapeutics (Vijayaraghavan and Kamala Nalini, 2010).

Nanotechnology presents potential opportunities to create

better materials and products. Silver nanoparticles (AgNPs) have been

extensively used in various areas like food service, medical instruments,

personal care products, solar energy conversion, building materials,

water treatment, catalysis and textiles because of their antibacterial

effect. Optoelectronic, physicochemical and electronic properties of metal

nanoparticles are determined by their size, shape and crystallinity.

Therefore, the synthesis of monodispersed nanoparticles with different

sizes and shapes has been a challenge. The seaweeds are rich in

biologically active substances that may reduce the silver nitrate and

hence biosynthesis of nanoparticles using seaweeds has turned much

attention towards the utilization of renewable marine resources

(Kumar et al., 2012b). However, few reports are there regarding the

usage of seaweeds in the green synthesis of silver nanoparticles

(Nabanita et al., 2009; Devina Merin et al., 2010; Swaminathan et al.,

2011; Mahdieh et al., 2012; Murugesan et al., 2011; Suriya et al., 2012).

Biological synthesis of nanoparticles is a relatively new

emerging field of nanotechnology, which has economic and eco-

friendly benefits over chemical and physical processes of synthesis.

The brown marine algae Sargassum muticum aqueous extract was used

as a reducing agent for the synthesis of nanostructure silver particles

(Ag-NPs). Structural, morphological and optical properties of the synthesized

nanoparticles have been characterized systematically by using FTIR,

XRD, TEM and UV–Vis spectroscopy (Susan Azizi et al., 2013). The

nanoparticles were not chemically bonded to the substrate; however,

we found out that the sample was stable during investigation in the

air which allowed us to measure the size distribution of nanoparticles.

In order to minimize the effect of AFM tip radius in the measurements,

a height of the particles has been measured (Ebenstein et al., 2002).

Biosynthesis of silver AgNPs from seaweed extracts is

currently under exploitation. The synthesis of AgNPs from silver precursor

and silver nitrate using an aqueous extract of seaweed Gracilaria

corticata are cost effective, eco-friendly and thus can be an economical

and efficient alternative for large-scale synthesis of nanoparticles. The

organic compounds present in the filtrate of G. Corticata were mainly

responsible for the reduction of silver ions to AgNPs. The filtrate when

added to 1 mM aqueous silver nitrate solution at 121°C changed to

dark brown colour solution within ten minutes, which confirms the

bioreduction. These extremely stable AgNPs were characterised by UV-

Vis spectrophotometer, FTIR, XRD, TEM, and EDAX analysis. The

nanoparticles exhibited maximum absorbance at 424 nm in the UV

spectrum. The presence of proteins was identified by FTIR. TEM

micrograph revealed the formation of polydispersed and spherical

shaped nanoparticles with the size range of 10-50 nm and the presence of

elemental silver were confirmed by EDAX analysis. These nanoparticles

showed cytotoxic activity against Hep2 cells (Saranya Devi and

Bhimba, 2014).

In this intensive investigations were made on the potential

of Seaweed Liquid Fertilizer (SLF) obtained from the U. reticulata,

S. wightii and H. musciformis on the germination, biochemical constituents

and growth parameters of black gram (Vigna mungo L.) under laboratory

and in field conditions. We had seen the biochemical constituents,

growth and yield parameters were significantly increased in SLF

treated black gram.

Extracellular synthesis of silver nanoparticles by U. reticulata,

S. wightii and H. musciformis extracellular synthesis of AgNPs has

been monitored using UV–Vis spectrophotometer, the protein-AgNPs

interaction examined by FTIR, the crystalline nature of AgNPs studied

by X-Ray diffraction, size and morphology of the AgNPs analysed using

SEM, AFM and their antibacterial effects against some selected human

pathogens were reported and photocatalytic degradation of methyl

orange using Silver nanoparticles synthesized from seaweeds.