A Review on plant Nutrients from Freshwater Algae Koffi Tindo Kevin1

A Review on plant Nutrients from Freshwater Algae
Koffi Tindo Kevin1, Dr. Sumit Kumar2, Prof. Dhamesh H. Sur3
Bachelor of Engineering, Gujarat Technological University,
Biotechnology Engineering Dept., V.V.P. Engg. College, Rajkot, 360005, Gujarat, India
[email protected]
Different types of macro- and micro-nutrients are required by agricultural and horticultural plants for their normal growth and development. Unfortunately often these nutrients are not available in sufficient amount in different soils, resulting in an abnormal growth and development of the plants. Alternatively Algae, a simple, aquatic plant-like organism possesses extraordinary features that can remediate the lack of nutrients in plants. Such plant nutrients can be isolated from various kinds of freshwater algae in an economic manner. The present project work aims at collecting locally available freshwater algae, isolating plant specific nutrients from them and formulating a product which would be utilized as organic fertilizer for majority of agriculturally important plants. The development of this kind of product could help curtailing the use of chemical fertilizers and thereby improving the health of the environment.
Keywords: Macro-nutrients, Micro-nutrients, Freshwater, Algae, Plants
INTRODUCTION

Plant nutrients are the chemical elements necessary for plant health and its metabolism. Plant nutrients are naturally found in the soil. The plant nutrients content available to soil varies from soil to soil in quantity as well as in composition. This variation or lack of certain nutrients in certain soils, leads to a restriction in certain plant growth and poor productivity. The search for solution to eradicate these problems gave birth to the use of chemical fertilizers. Chemical fertilizers are defined as inorganic material of wholly or partially synthetic origin that is added to the soil to sustain plant growth. The particularity of chemical fertilizers is that nutrients are readily available to the plants and immediate improvement occurs in days (Julie day, 2017). Unfortunately, despite its success, chemical fertilizer is limited to the growth of plant and do not sustain the soil fertility and it is also associated with some environmental problems affecting obviously living beings.
These limitations give importance to the alternative source of both soil and plant nutrients. These sources are found to be bio fertilizers and organic fertilizers.
As the name suggests, bio fertilizer is defined as a preparation that contains living cells or latent cells of efficient strains of microorganisms capable to help the crop plants uptake of nutrients by their interactions in the rhizosphere when applied through seed or soil (Dr. Tohid Nooralvandi, 2016). While in terms of fertilizer, organic means a product that has been minimally processed and the nutrients remain bound up in their natural forms rather than being extracted. Organic fertilizers require microorganisms to break down the compost or manure according to the rule of the nature and this retards the release of nutrients to the soil as well as plant…The examination of the limitations of both chemical and organic fertilizers gives sense to the current review titled PLANT NUTRIENTS FROM FRESHWATER ALGAE.

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Algae are living organisms containing plant nutrients. They are defined as any of the several divisions of simple photosynthetic organisms, especially certain thallophytes, variously one-celled, colonial, or filamentous, containing chlorophyll and other pigments and having no true root, stem, or leaf. Algae occur in various shapes and sizes and have different ecological roles. Thousands of species of algae occur worldwide in both freshwater and marine water. The main different types of algae found in freshwater are the green algae or Chlorophyta, red algae or Rhodophyta, blue-green algae or Cyanobacteria and diatoms or Bacillariophyta.

Ecologically, algae are primary producers as they fix carbon and generate biomass; they are one of the three major groups of photosynthetic organism within the freshwater environment.
Also, many of Algae species contains all the nutrients necessary for the growth and development of terrestrial plants.

Algae play crucial roles in the environment; they produce oxygen as a waste product of photosynthesis, algae and especially Cyanobacteria fixes nitrogen from the atmosphere and makes it available for the nutrient cycles, they help to purify water by absorbing nutrients and heavy metals from streams and rivers, they are also known to be valuable indicator for environmental quality.
Due to their several potentialities, algae are used in number of industries to commercially produce useful end products such as foods, pharmaceuticals and medicines, animal feeds, fuels and fertilizers.

In addition to their potentialities, algae are easily available in the nature and can be found in habitats such as freshwater (ponds, streams, wetlands, etc.), saltwater (sea, marine algae), and area containing moisture or water. The growth of algae in freshwater reflects the possibility to use wastewater (from industries or home) for algal growth. In this way, algae are beneficial in both environmental cleaning and biomass production (including plant nutrients). Also, the growth of algae in saltwater implies that they do not necessary require freshwater to be cultivated, and hence do not competing with crops for agricultural land. Hence, algae can constitute a potential economical source in the field of agriculture (biofertilization).

Many researches have been performed on algal biofertilization; these earlier researches have been achieved by using algal extract in combination with commercially chemical nutrients. For example, Clayton Beaty has formulated a fertilizer comprising urea, ammonium phosphate, muriate of potash, magnesium sulfate, fish soluble, and extract of seaweed (Clayton Beaty, 1997). Although this method uses chemical to fasten the uptake of nutrients and also seawead extract to improve the soil and plant quality, it does not ensure the safety of crops and the soil.
Although it is more smarter and even imperative to find and develop an alternative and sustainable way to improve agriculture without affecting the environment and the fertility and friability of the soils, but rather to fasten the growth of plants healthly and economically.

Hence, this current research aims at formulating a pure algal biofertilizer from dried green macro algal samples. This work will therefore consist of centrifugation, nutrient analysis and quantification and application to plants.
Plant nutrition

2.1- Plant nutrients, importance, and deficiency symptoms
2.2.1- Macro-elements- Primary Nutrients:
Nitrogen (N), Phosphorus (P) and Potassium (K) form the primary nutrients of the macro-elements. They are usually called NPK and are required in large quantity. They should be readily available to the soil.

Table 1: Primary Nutrients, their functions and Deficiency Symptoms
Nutrients Available to soil as Functions Deficiency symptoms
Nitrogen
(N)
Nitrate
(NO3-)
.Ammonium
(NH4+) An important part of chlorophyll which responsible of the light absorption during photosynthesis.

Formation of protoplast and plant growth regulators (amino acids, enzymes, vitamis, etc.).

Improve the quantity and quality of dry matter and proteins in vegetables.

Probability of stunted growth in plant due to the lesser cell division.

Occurrence of chlorosison the older leaves.

Reduction in flowerin and pre-maturation in some crops.

Phosphorus
(P) Orthophosphate
(HPO42- and H2PO4-) Energy storage and transfer (ADP, ATP, DPN, and TPN) during photosynthesis and respiration.

Constituent of RNA and DNA.

Needed for the formation of seeds and it is also largely required in shoots and root tips to support the rate of metabolism and cell division.

Helps in development of root, flower and seed. Also it reduces the disease effect in some plants.

Improvement of the crop quality.

Slow, weak, and stunted growth.
Dark to blue-green coloration on older leaves of some plants.

Purpling of leaves and stems.
Delayed maturity and poor seed and fruit development.

Potassium
(K) Potassium ion (K+) •Activates enzyme to promote metabolism.

Controls the opening and closing of leaf stromates.

•Maintaining the balance of electrical charges at the site of ATP production during photosynthesis.

•Translocates photosythates such as sugars for plant growth or storage in fruits or roots.
• Participates in ATP and proteins synthesis.

•Improve the resistance to disease, grain and seed size,and the quality of plants. •Occurrence of chlorosis
• Slow and stunted growth.
• In some crops, stems are weak and lodging
• Reduction in the size of seeds and fruits and yield.

2.2.2 SecondaryNutrients:
The secondary nutrients are Sulfur (S), Calcium (Ca), and Magnesium (Mg). they are required in relatively lesser quantity than primary nutrients. The quantity needed for the growth of plants may be easily obtained from soil, but the use of fertilizer may help in boosting the yield and hence the economic.
Table 2: Secondary Nutrients, their functions and Deficiency Symptoms:
Nutrients Available to soil as Functions Deficiency symptoms
Calcium
(Ca) Calcium ions
(Ca2+) • Formation of the cell wall membrane and its plasticity,
• Activator of several enzyme systems during protein synthesis and transfer of carbohydrate.

• Detoxification of the plant.

• Production of seed in peanuts.
• Reduction of acidityof soils, when they are lime • Brown coloration and death of the plant.

• Limitation in growth,
• Newly growing leaves may stick together at the margins to causes tearing and unfurl, and weakness in the structure of the stem.

•Cupping and Crinkling of younger leaves and deterioration of the terminal bud.
Magnesium
(Mg) Magnesium ions
(Mg2+) Major constituent of the chlorophyll molecule and it is therefore actively involved in photosynthesis.
• Co-factor in several enzymatic reactions enabling the activation of the processes of phosphorylation.

• Stabilization of ribosome particles and the structure of nucleic acids.
• Mg assists the movement of sugars within a plant • Occurrence of chlorosis in older leaves.
• Drop of younger leaves
Sulfur
(S) Sulfate ions
(SO42-) Constituent of certain amino acids.
• participates in the metabolism of B vitamins biotin and thiamine and co-enzyme A.
• formation of chlorophyll,nodule in legumes, and protein structure stabilization Occurrence of chlorosis in both younger and older leaves.

However, deficiency is not commonly found in most plants.

• Retardation of growth rate.

•Thin, stiff, and woody plant stems.

2.2.3- Micro-nutrients:
The micronutrients are Zinc (Zn), Iron (Fe), Boron (B), Chlorine (Cl), Manganese (Mn), Copper (Cu), Sodium (Na), Colbalt (Co), Molybdenum (Mo), Silicon (Si), and Vanadium (V). these micronutrients are required in quantity lesser than that of secondary nutrients. Generally they concentrations in plant are below 100 ppm (parts per million) level.

Nutrients Available to soil as Functions Deficiency symptoms
Boron
(B) Borate
(H3BO3) Involved in RNA, seed and cell wall formation, cellular and enzymatic activities, pollen germination, pollen tube growth, lignin synthesis and in root growth promotion. Appearance of stunted growth, thickened, curly and brittle leaves, hollow and black hearts, crooked and cracked stem, distorted and lumpy fruit, calyx splitting, midribs crack, brown colour (in Chinese cabbage), and pith in hollow stem.

Copper
(Cu) Ion Cu++ Involved in synthesis of the chloroplast proteinplastocyanin, stability of chlorophyll and other pigments. Reduction in growth, distortion of the younger leaves, necrosis in the apical meristem, bushy appearance in trees,bleaching in young leave,defoliation and dieback of twigs, stunted and chlorotic plant.

Iron (Fe) Fe2+
Fe3+ Involved in plantand protein metabolism (photosynthesis and respiration).
• Part of protein ferredoxin and is required in nitrate and sulfate reductions.

Involved in the synthesis and maintenance of chlorophyll in plants. Interveinalchlorosis in younger leaves. The youngest leaves maybe white, because Fe, like Mg, is involved in chlorophyll production. • Usually observed in alkaline or over-limed soils.

Chlorine
(Cl) Chloride ion (Cl-) Involved in evolution of oxygen during photosynthesis,
•Increases osmotic pressure of the cell and the water content of plant tissues.

• Fights against fungal diseases Occurrence of chlorosis of younger leaves and wilting of the plant.
Zinc
(Zn) Zn ++ Involved in synthesis of tryptophan, metallo-enzymes, activation of carbonic anhydrase, and RNA and protein synthesis.

Occurrence of Interveinalchlorosison younger leaves, stunted growth, and
reduction in fruit formation.
Molybdenum
(Mo) Molybdate(MoO4) • Component of both the enzymes nitrate reductase and nitrogenase necessary for the assimilation of N in during nitrogen fixation.

Occurrence of chlorosis in older and middle leaves, stunted plant, and restriction in flower formation.

Manganese
(Mn) Mn2+, Mn3+ Involved in the primary function of metabolism as a part of plant enzyme system, pyruvate carboxylase formation, oxidation-reduction process during photosynthesis, indole acetic acid oxidase activation. Occurrence of chlorosis in young tissues.

Importance of Algae in Agriculture
With the rise in human population, it is becoming imperative to focus on sustainable agriculture.

The use of algae as source of fertilizer is an alternative and sustainable way to improve agriculture without affecting but rather increasing the health and productivity of the plant as well as the soil. The success of algae as bio fertilizer lies on its biological importance.

In agriculture, algae play a crucial role in arid and semi-arid ecosystems. Also their distribution in the environments constitutes a mean of indication of the pollution state of these environments. In addition, algae constitute an alternative substituent of the chemical soil conditioners and hence, they improve the properties of both plants and soils. For instance, the blue green algae also called cyanobacteria are a type of microalgae responsible for the photosynthesis of microbes in the soil. Furthermore, they are found to play very important roles in both the fertility and reclamation of the soil. When used in agriculture, blue green algae ingest organic acids capable to provide phosphorus while increasing its uptake by the plant; cyanobacteria provide biological nitrogen to the plant through the process of biological nitrogen fixation and therefore increase the organic matter of the soil. Many plant growth regulators such as amino acids, vitamins, polypeptides, antifungals, and polymers (exopolysaccharides) that helps to improve the structure of the soil and exoenzyme activity of the plant. Even the metal irons present in the environment can be cleaned up by sequestering them in their cell membrane. And finally algae are capable to effectuate crust formation and stabilization of soil aggregation. (Abdel-R. et al, 2010).

The preference of bio fertilizer over chemical fertilizer stands as the result of many experiments. Bio fertilizers are not only environment friendly but also, they ameliorate the physical property of the soil, increase the water retention capacity, avoid nutrient depletion, and enhance the value of the growing crop. S. Sujanya and S. Chandra (2011), have proved that the combination of 3:1 FYM: NPK ratio gives a better yield in crop and also in soil and protein content of the crop than using NPK alone as chemical fertilizer. They also proved that the use of biological mixture of BGA, Azosporillum and Azotobacter with respect to 3:1 combination improve the nutritive value of the crop leading to an increase in its commercial value.

Even when used in marginal farming, algae based fertilizer helps to render the soil vital and in case of rice farming, it can even extend the cultivation of rice to a longer time.(S. K.Goyal, 1993).

Algalization is BGA bio fertilizer used in rice, it is an environment and eco-friendly agro-ecosystem when used in paddy cultivation and also reduces energy input. This fertilizer also helps to stabilize the soil, release growth promoting substances, add organic matter, solubilize the insoluble phosphate and finally improve the physico-chemical properties of the soil (D. Shu, 2012).

The whole algal biomass is rich in certain useful constituents including vitamins, enzymes, specific organic substances, minerals, and metabolites and this has necessitated a developed process for a greater need of these compounds for various uses (Anil Kumar Sing et al., 2016).
According to Arif et al. (2006), when micronutrients are applied to the soil, they may not meet the requirements of the crop for the growth and nutrient balance within the plant tissue, hence an alternative effective way is to use these micronutrients in form of foliar spray. It is in the same objective Mahmoud M.Shaaban et al. (2010), have studied the effect of foliar spraying with the green microalgae Scenedesmus sp. using water extract and or micronutrients commercial compound and their combinations on the nutrient balance and dried weight accumulation in wheat plants. After this study, Mahmoud M. Shaaban et al. have found that the extract of algae can partially substitute micronutrient foliar fertilizer and is crucial for the growth of wheat plants in the nutrient balance within the tissue of the plant.

In the same objective, Clayton Beaty has formulated a fertilizer comprising urea, ammonium phosphate, muriate of potash, magnesium sulfate, fish soluble, and extract of seaweed (Clayton Beaty, 1997); but such a method using chemicals may decrease the fertility of the soil in future and even increase the production cost, although the method uses also seaweed extract, it does not ensure the safety and the sustainability of the plant and the soil. Therefore, Algae a simple, aquatic plant-like organism possesses extraordinary features that can improve the quality of fertilizer.

In a review journal of Biotechnology by Abdel-Raouf et al. (2012), it has been tried to demonstrate the important characteristics of algae in agriculture and more precisely importance of microalgae in agriculture. In this review (Abdel-Raouf et al. 2012), it has been resumed that algae is capable to increase the organic matter content of soil, capable to release organic acids that can increase the phosphorus content in the soil as well as its uptake by plant, capable to produce and release bioactive extra-cellular substances that can influence the growth and development of plant, also algae are capable to concentrate ions of metal present, capable to capture nitrogen through nitrogen fixation, capable to form crust and stabilize soil aggregation by mean of the extra-cellular polysaccharides of soil aggregate.

To exploit precisely these tremendous characteristics of algae, it is very important to study their different groups, species and characteristics. It is then in this objective that, an article by “The Age of Aquarium” (2017), has classified freshwater algae into. In the same objective, Powell et al. (2014), have developed a composition and methods to collect algae in order to exploit its useful characteristics.

Classification of algae
Algae are broadly classified into microalgae and macroalgae on the base of their size and morphology.
Microalgae are classified into chlorophyta, rhodophyta, haptophyta, stramenopiles and dinophyta.

Table 4: Classification of Microalgae.

Chlorophyta Rhodophyta Haptophyta Stramenopiles Dinophita
Habitats Freshwater, marine, terrestrial environment Mostly marine,freshwater Mostly marine and few in freshwater Marine, freshwater and terrestrial environment Marine and freshwater
Cell types Unicellular and multicellular Multicellular and unicellular Unicellular and colonial Unicellular and colonial Unicellular
Pigments
Chlorophylls a & b Phycocyanin and phycoerythrin Mode of nutrition Photoautotrophic Heterotrophic and photosynthetic
Common species Chlorella vulgaris, dunaliella salina, haematococcus pluvialis Crypthecodinium cohnii,
Mode of Reproduction Vegetative Sexual
Mode of movement Flagella Flagella Flagella
2- Macroalgae
Macroalgae are classified into 4 phyla., such as rhodophyta, ochrophyta, chlorophyta, anf cyanobaceria.

Table 5: Classification of macroalgae.

Rhodophta (red algae) Ochrophyta (brown algae) Chlorophyta (green algae) Cyanobacteria (blue green algae)
Habitats Mostly in marine Marine Freshwater and marine Cell types Unicellular and multicellular Unicellular and multicellular Uni and multicellular Pigments
Phycoerythrin, chlorophyll a & b, and carotene Carotenoid, fuxoxanthin and chlorophyll a & b Chlorophyll a & b Mode of nutrition Photosynthetic Photosynthetic photosynthetic Common species Mode of Reproduction Asexual Sexual asexual, and vegetative Sexual and asexual Motility Flagella Flagella and non flagella Collection of freshwater algae sample for nutrient analysis
The day times affect the position of algae in the water. In morning, they move towards the surface and then sink slightly to the the lower region of water in afternoon tome. Hence, collection should occur preferably between 8:30 am and 12: 00 pm (Asma Manzoor etal., 2012).

5.1- microalgae collection
Freshwater microalgae can be collected from various sources. These sources are large and small rivers, lake, ocean, and stream.
Equipments: the collection of microalgae samples for nutritional analysis requires following materials: a pole type sampler or horse-pipe sampler, or a depth sampler. Other materials such as gloves, sample bottles, Lugol’s solution and pipette, a cool box containing ice bricks, and a bucket (5L) freshwater for washing hands and materials are also used.

Whatever the selected source or type of algae, following procedures are preponderant in algal sample collection. It is important to select a representative site of collection for ecological surveys, all the the physical characteristics of the algae present in the selected site should be noted. These characteristics might be the colour, odour, presence of dead organisms or other wastes. The sample bottles should be labeled with a water proof, addition of few drops of Lugol’s solution is important to preserve the sample and finally the collected samples should be kept in a cool box containing ice briks.

(Department of environment and heritage protection, May 2017).

5.2- macroalgae collection
According to Bristish phycological society, macroalgae can be collected by hand in shallow water and by snorkeling in deeper water. Although, some materials such as glass bottomed box, three-prondeg granel or drege can be used. After collection the sample should be squeezed in order to remove the water before storage.

Extraction of active nutrients
Extraction of nutrients for plant growth purpose consists of extracting biological compounds also called biostimulants from a prepared sample of algae. This requires various pre-treatment steps such as homogenization, treatment with acids and alkalis, freezing, and micronisation are carried out. The homogenization or washing process is useful because it helps to improve the extraction efficiency by increasing the degree of the active ingredients present in the sample. Acids and alkalis are used to weaken the algal cell wall in order to ease the extraction. Drying (freezing) is a mean to disrupt the cell wall of certain species of algae, so sufficient knowledge about the species under analysis is imperative for selecting the suitable drying method. Micronisation (or grinding) is used to increase the extraction rate by improving the surface area. But the micronizer should not be over heated to lose active ingredients. These pre-treatments are related to some controllable parameters.

Table 6: controlled parameters and their roles
Parameters Roles
Ph Alkali pH reduces the surface tension, remove partially lignin, remove completely acetyl or uranic ester group of hemicellulose
Change the biochemical solubility
Time Biochemical extractability
Long time of extraction can lead to microbial growth and oxidation of phenolics resulting in lesser yield.

Type of solvent For promoting mass transfer in order to increase the rate of extraction
Acidic solvent also extends the degradation of cell wall
Minimum quantity of solvent capable to attain diffusion should be taken into account to avoid high cost
Selection of solvent-algae ratio, algae-solvent ration and algae particles size are also important.

Agitation Temperatue Biochemical extractability
Increase analyte solubility
Increase diffusion rate
Decrease viscosity
Decrease solution surface tension
6.1- washing
Washing of algae consists of removing all the visible unwanted debris contained in the sample by using tap water followed and removal of the extraneous particles by a repeated washing with deionised water.
6.2- Drying
The drying process of algae sample is crucial in the extraction process. It affects not only the yield but also the extraction cost. Several methods such as sun drying, oven drying, freeze drying, spray drying, roller or drum drying, and fluidized-bed drying are used to dry algae.
Drying of macroalgae can be achieved by the above first three methods (sun drying, oven drying, and freeze drying). In a comparative study on the effect of these three drying methods on the nutritional composition of the macroalgae species Sargassum hemiphyllum, Chan et.al (1997) have scouted that the oven and freeze dried samples showed higher ash content than that of sun dried and thus, the mineral (nutrients) content of sun dried sample were also lesser compared to others. Except the calcium content that is much higher in sun dried sample than oven and freeze dried sample. This was probably due to over exposure to air and leaching of nutrients. In other hand, the over drying method was shown to be not only the fastest method but also the one showing yield of sodium (Na), potassium (K), iron (Fe), zinc (Zn), copper (Cu), manganese (Mn), and aluminum (Al).

Regarding microalgae drying, during a study of the dewatering and drying methods for microalgae Ching-Lung Chen et.al ( 2015) have scouted the characteristic of the commonly used dewatering and drying technologies of microalgae. In this study, they concluded that the methods of drying and even the dewatering process depend on the properties of the microalgae under study, the required design and the related capital cost. Hence for microalgae, sun drying, spray drying, freeze drying, roller or drum drying, and fluidized-bed drying are used. Sun drying requires larger area even if it is not costly, while spray and convective drying methods tends to alter the nutritional composition of microalgae. As for freezing, nutrients are preserved but high operation cost is its disadvantage.

6.3- grinding and ash content
Grinding does not affect the nutritional composition of algae, but raise in temperature of the grinder can lead to loss of nutrients. Grinding is important because the decrease in size of algae sample leads to an increase in surface area causing high rate of extraction. The dried weight of the sample should be taken before grinding.
The amount of ash is directly proportional to the amount of nutrients present in the sample. Higher the weight of ash, higher the amount of nutrients are available. Following formula can be used to determine the amount of ash in percentage.

% Ash=Mass of ashMass of dried sample×1006.4- Extraction methods
Generally solvent extraction methods are used in extraction of algae components. Although various methods such as microwave assisted extraction, enzyme assisted extraction, supercritical fluid extraction using CO2 as solvent, ultrasound assisted extraction and soxhlet extraction can also be used to extract active nutrients from algae. Unfortunately these techniques are not only costly but also toxic to environment due to the toxic solvent associated.
Environment friendly methods developed by Godlewska et.,al (2016) show may advantages. These methods are boiling and soaking of algae extract (pre-treated sample). Through these methods, various macro and micronutrients are characterized. In addition, substances such as lipids (n-3 and n-6) fatty acids), polyphenols, and some antibacterial properties for Eschericha Coli and Staphylococcus aureus can also be characterized.
The boiling method was performed by adding 50g of the prepared sample into a 250ml flask containing 150ml of distilled water. The whole is boiled by mean of water bath for 30 min and then centrifuged at 4250 rpm for 5 min. After centrifugation, the sample is then filtered with a Whatman no1 filter paper, and the obtained supernatant represents 100 % algal extract ready to undergo nutrient analysis.
In the soaking method, the whole (sample + 150 ml of distilled water in 250 ml flask) is kept for two days instead of boiling, and then follows the same procedure from centrifugation. Godlewska et. Al found that the boiled extract was rich in phosphorus (P), sulfur (S), and boron (B) while the soaked extract was rich in calcium (Ca), magnesium (Mg), and iron (Fe).
In addition, Asma Manzoor et al., have extracted nutrients from seaweed by hot and cold water method. In this method, the prepared sample was firstly homogenized by mean of deionised and distilled waterin a blenderfor 10 min and the PH is adjusted by mean of hydrochloric acid drops. The sample was again homogenized for 5 min and followed by neutral PH adjustement. The whole is then boiled at a temperature between 28 to 100 0C for 3 h and filtered through a muslin cloth followed by whatman gap 1 filter paper. The obtained filtrate is then ready for undergoing nutrient characterization.

Nutrient analysis methods
Various methods are available for characterizing algae plant essential nutrients in algae sample. The most commonly used methods are kjeldajl techniques, photometry and spectroscopy methods.

Kedjal method is used to estimate nitrogen content (Rober Banks, 1903). The method consists of heating the algal sample with sulfuric acid to liberate the ammonium of sulfate (reduced nitrogen) and adding sodium hydroxide to convert the ammonium of sulfate obtained into ammonia which is then titrated to obtain the amount of nitrogen and ammonia present (Asma Manzoor et al., 2012 )
Photometry methods are used to determine phosphorus, iron, manganese, zinc, copper and sodium. The spectro-photometrical method using molybdate-vanadate technique permits the determination of phosphorus content in algae sample. Flame photometry is used to determine potassium content while Atomic Absorption spectro-phtotometry(AASP) is used to quantify various nutrients such as Iron, Manganese, Zinc, Copper, and Sodium.

In addition, spectroscopy methods are also used in plant nutrient analysis. These methods include Atomic spectroscopy techniques such as Inductive Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) enabling the measurement of calcium and magnesium content and Flame photometry Atomic Emission Spectroscopy (F-AES) for determination of sodium and potassium content. Optical spectroscopy method like inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) is widely used for determining all plant essential nutrients content in algae sample.
Whatever the methods used, the target of characterizing actives nutrients or plant nutrients in algae extract, for biofertilization purpose, to obtain not only sufficient amount of nutrients but also in suitable ratios (Asma Manzoor et al., 2012).
Conclusion
The nutritional requirement of plants for their growth and metabolism ranges from micro to macro nutrients in terms of required quantity but not the importance. However all the nutrients are crucial in the development of plants. The lack of appropriate amounts or ratios of these nutrients can be observed on the morphology of the plant and hence leads to abnormal growth and poor yield. The importance of algae in agriculture lies in the facts that, algae can not only provide all the plant essential nutrients in form of bioactive nutrient readily available to plant like inorganic nutrients but also can supply important growth regulators capable to increase the economical value of plants. The knowledge on the diverse nature of algae is preponderant in the discrimination and/or improvement of the compatible of algae species for agriculture. The collection and preservation procedures affect not only the extraction process but also the cost of productivity. Many extraction techniques are available among which water solvent extraction seems to be the most simplest and less cost. In order to confirm and ameliorate the suitability of algae extract, nutrient analysis must be performed. The commonly used technique is Inductive Coupled Plasma Optical Emission Spectroscopy (ICP-OES) capable to quantify all the essential nutrients present and also other growth factors available in the extract.
The availability of freshwater and the fast and easy growth of algae constitute good factors to promote algae based biofertilizer and mitigate the use of chemical inorganic fertilizer. Although further studies must be considered in order to ease the access of nutrients analysis processes to help farmers go green.
REFERENCES