*In vitro* **Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants**

A. Bakrudeen Ali Ahmed, S. Mohajer, E.M. Elnaiem and R.M. Taha

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50690

## **1. Introduction**

254 Plant Science

[40] Premkumar A, Mercado JA, Quesada MA. Effects of *in vitro* tissue culture conditions and acclimatization on the contents of Rubisco, leaf soluble proteins, photosynthetic

pigments and C/N ratio. Journal of Plant Physiology 2001;158 835-840.

Tissue culture has been applied to diverse research techniques such as viral elimination, clonal propagation, gene conservation, *in vitro* fertilization, mutation, induction for genetic diversity, genetic transformation, protoplast isolation and somatic hybridization, secondary metabolite production and other related techniques. The commercial production of ornamental plants is growing worldwide. Its monetary value has significantly increased over the last two decades and there is a great potential for continued further growth in both domestic and international markets. About 156 ornamental genera are propagated through tissue culture in different commercial laboratories worldwide. About 212.5 million plants including 157 million ornamental plants amounting to 78% of the total production were reported [1]. These plants are over exploited due to their high medicinal value and hence, propagation of the plants by tissue culture may be mandatory, which offers a greater potential to deliver large quantities of disease-free, true-to-type healthy stock within a short span of time. Biotechnological interventions for *in vitro* regeneration, mass micropropagation and gene transfer methods in forest tree species have been practiced with success, especially in the last decade. Against the background of the limitations of long juvenile phases and lifespan, developments of plant regeneration protocols of ornamental species are gaining importance. Ornamental industry has applied immensely *in vitro* propagation approach for large-scale plant multiplication of elite superior varieties. During *in vitro* condition, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. After the plantlets reached optimum growth in the culture containers after a certain growth period, it can be transferred to *ex vitro* condition to allow continuous growth of the plantlets. As a result, hundreds of plant tissue culture laboratories have been set up worldwide, especially in the developing countries due to cheap labour costs.

© 2012 Ahmed et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Ahmed et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Plant tissue culture media is normally rich in sucrose and other organic nutrients that can support organogenesis in plants but also the growth of many microorganisms (like bacteria and fungi). To overcome and prevent contamination in media preparation, sterilization should be done thoroughly. Sterilization of nutrient media can be done in an autoclave (large pressure cooker), less often by filtration and seldom by irradiation [2]. The container with the medium should be properly closed and autoclaved at 121ºC, 105 kPa, for 20 minutes. It also identified that good sterilization relies on time, pressure, temperature and volume of the object to be sterilized [2]. The sterilized nutrient media should be stored in a sterile box that has previously been disinfected with 70% alcohol [2]. Some of the plant growth regulators such as giberellic acid (GA3), zeatin, abscisic acid (ABA), urea, certain vitamins, pantothenic acid, antibodies, colchicines, plant extracts and enzymes used in tissue culture is thermolabile. These compounds should not be autoclaved and filtersterilization is often used if a thermolabile substance is needed in a nutrient medium.

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 257

callus; promote rooting, in the conventional cutting propagation and in organized tissue culture. Auxins can also promote plant sex differentiation, the formation of flowers and to promote the formation of seedless fruit. Auxins main use in tissue culture to induce callus and, also they are important use is in line with a certain amount of cytokinin for root differentiation, lateral bud germination and growth, as well as in certain plants induce embryogenesis. Since, many research articles have dealt at great length about the status, applications, potentials and needs in tissue culture of ornamental plants. Since, the major effects have been made to develop basic back ground technologies for consistent production and regeneration of calluses from diverse group of medicinal plants [3]. The techniques which have been so far described for propagation of ornamental plants through tissue culture have been tested on the laboratory scale and have not been validated for their suitability in commercial scale production. However, the following aspects have to be critically studied if the economic prospects associated with *in vitro* culture technology are to be realized. Generally, the application of plant cell and tissue culture technique which is commonly known as *in vitro* cloning can also be divided into several procedures, including meristematic cultures, vegetative explant cultures, callus induction, suspension cultures, direct and indirect somatic embryogenesis, synthetic seed production, *in vitro* flowering, *in vitro* mutation breeding, protoplast and also somatic hybridization process. Some of these techniques would selectively applied to selected plants overcome generation incapability of the plants. This *in vitro* technique is very useful in ensuring sustainable, optimized sources of plant-derived natural products. However, *ex situ* cultivation should be preceded by proper evaluation of the plants for their ability to produce the required bioactive constituents before commencing cultivation or introducing the technology to potential growers. The ability of plants to produce certain bioactive substances is largely influenced by physical and chemical environments in which they grow. Plants also produce certain chemicals to overcome abiotic stresses. In this aspects plant tissue culture developed callus influenced by medium, explants, plant growth regulators, color lights, temperature, photoperiod and carbon sources are helpful to produce valuable secondary metabolites compounds in many studies [3,4]. Growing a plant outside its natural environment under ideal conditions may therefore, result in being unable to produce the desired bioactive substances, hence the need for prior

Ornamental plants are used especially as decorative houseplants and for landscaping. Ornamental plants are unique for their sheer beauty and variety of leaves. For example Begonias, which have a medicinal value. It is a temperate plant, which is commercially used as a flowering pot plant. It does not produced seeds. It is a winter flowering plant and was developed from cross between other species of the same family. Begonia plant normally has thick, shiny, dark green leaves. Although Begonias can be readily vegetative propagated, they are susceptible to many pathogenic bacteria, fungi, and nematodes [5]. Nowadays, the seaweed market has grown as predicted with prospects to go even further. Therefore

evaluation.

**4. Research course** 

### **2. Problem statement**

*In vitro* plant tissue culture needs the formulation of a complete nutritional medium and for exploration of plant physiological processes; it needs the addition of effective plant growth regulators. These two aspects can be considered to plant tissue culture the wings to take off. With the starting of common or specific media and the selection of appropriate plant tissue culture, enable induction of cell division, callus growth, differentiation of shoots, roots and embryos. Commonly, synthetic analogues are used, mainly; 1-naphthalene acetic acid (NAA), indole butyric acid (IBA), 2,4-Dichlorophenoxyacetic acid (2,4-D) in plant tissue culture for the induction of plant cells and the regeneration of the root, bud, embryo-like body which has a high organizational structure of specialized cells. The main physiological role of cytokinins; promotion of cell division and expansion, buds induction, differentiation, promote lateral bud sprouting and inhibition of senescence. *In vitro* tissue or organ will be soon aging. Such as cytokinin treatment can slow the aging process. Another class of hormones used in tissue culture is gibberellins. The main physiological effects of gibberellin (GA) are buds induction. It is commonly used in plant tissue culture to promote the growth of the seedling stem elongation. It is also affect cell differentiation of cambium and it often works in synergy with auxins. In addition, the other hormone such as abscisic acid (ABA) is one of the plant growth substances; it can be biosynthesized from mevalonic acid. It can control water and ion uptake by roots and to promote the adventitious shoots and absorb and prevent the phenolic production. Whereas, the ethylene one of the gases plant hormone, it is moved by diffusion around the plant rather than translocation. It has stimulates the final stage of fruit development and flower fall. The main function of ethylene in plant tissue culture, it can stimulate the respiration, seed germination, peroxidase enzymes and regulates the level of auxins. The low concentration of ethylene induces the proper resistance to the developed plant.

#### **3. Application area**

The physiological role of auxins promote cell growth and cell division, induction of the injured tissue of one to several layers of cells recovering the ability to divide to form callus; promote rooting, in the conventional cutting propagation and in organized tissue culture. Auxins can also promote plant sex differentiation, the formation of flowers and to promote the formation of seedless fruit. Auxins main use in tissue culture to induce callus and, also they are important use is in line with a certain amount of cytokinin for root differentiation, lateral bud germination and growth, as well as in certain plants induce embryogenesis. Since, many research articles have dealt at great length about the status, applications, potentials and needs in tissue culture of ornamental plants. Since, the major effects have been made to develop basic back ground technologies for consistent production and regeneration of calluses from diverse group of medicinal plants [3]. The techniques which have been so far described for propagation of ornamental plants through tissue culture have been tested on the laboratory scale and have not been validated for their suitability in commercial scale production. However, the following aspects have to be critically studied if the economic prospects associated with *in vitro* culture technology are to be realized. Generally, the application of plant cell and tissue culture technique which is commonly known as *in vitro* cloning can also be divided into several procedures, including meristematic cultures, vegetative explant cultures, callus induction, suspension cultures, direct and indirect somatic embryogenesis, synthetic seed production, *in vitro* flowering, *in vitro* mutation breeding, protoplast and also somatic hybridization process. Some of these techniques would selectively applied to selected plants overcome generation incapability of the plants. This *in vitro* technique is very useful in ensuring sustainable, optimized sources of plant-derived natural products. However, *ex situ* cultivation should be preceded by proper evaluation of the plants for their ability to produce the required bioactive constituents before commencing cultivation or introducing the technology to potential growers. The ability of plants to produce certain bioactive substances is largely influenced by physical and chemical environments in which they grow. Plants also produce certain chemicals to overcome abiotic stresses. In this aspects plant tissue culture developed callus influenced by medium, explants, plant growth regulators, color lights, temperature, photoperiod and carbon sources are helpful to produce valuable secondary metabolites compounds in many studies [3,4]. Growing a plant outside its natural environment under ideal conditions may therefore, result in being unable to produce the desired bioactive substances, hence the need for prior evaluation.

#### **4. Research course**

256 Plant Science

**2. Problem statement** 

proper resistance to the developed plant.

**3. Application area** 

Plant tissue culture media is normally rich in sucrose and other organic nutrients that can support organogenesis in plants but also the growth of many microorganisms (like bacteria and fungi). To overcome and prevent contamination in media preparation, sterilization should be done thoroughly. Sterilization of nutrient media can be done in an autoclave (large pressure cooker), less often by filtration and seldom by irradiation [2]. The container with the medium should be properly closed and autoclaved at 121ºC, 105 kPa, for 20 minutes. It also identified that good sterilization relies on time, pressure, temperature and volume of the object to be sterilized [2]. The sterilized nutrient media should be stored in a sterile box that has previously been disinfected with 70% alcohol [2]. Some of the plant growth regulators such as giberellic acid (GA3), zeatin, abscisic acid (ABA), urea, certain vitamins, pantothenic acid, antibodies, colchicines, plant extracts and enzymes used in tissue culture is thermolabile. These compounds should not be autoclaved and filtersterilization is often used if a thermolabile substance is needed in a nutrient medium.

*In vitro* plant tissue culture needs the formulation of a complete nutritional medium and for exploration of plant physiological processes; it needs the addition of effective plant growth regulators. These two aspects can be considered to plant tissue culture the wings to take off. With the starting of common or specific media and the selection of appropriate plant tissue culture, enable induction of cell division, callus growth, differentiation of shoots, roots and embryos. Commonly, synthetic analogues are used, mainly; 1-naphthalene acetic acid (NAA), indole butyric acid (IBA), 2,4-Dichlorophenoxyacetic acid (2,4-D) in plant tissue culture for the induction of plant cells and the regeneration of the root, bud, embryo-like body which has a high organizational structure of specialized cells. The main physiological role of cytokinins; promotion of cell division and expansion, buds induction, differentiation, promote lateral bud sprouting and inhibition of senescence. *In vitro* tissue or organ will be soon aging. Such as cytokinin treatment can slow the aging process. Another class of hormones used in tissue culture is gibberellins. The main physiological effects of gibberellin (GA) are buds induction. It is commonly used in plant tissue culture to promote the growth of the seedling stem elongation. It is also affect cell differentiation of cambium and it often works in synergy with auxins. In addition, the other hormone such as abscisic acid (ABA) is one of the plant growth substances; it can be biosynthesized from mevalonic acid. It can control water and ion uptake by roots and to promote the adventitious shoots and absorb and prevent the phenolic production. Whereas, the ethylene one of the gases plant hormone, it is moved by diffusion around the plant rather than translocation. It has stimulates the final stage of fruit development and flower fall. The main function of ethylene in plant tissue culture, it can stimulate the respiration, seed germination, peroxidase enzymes and regulates the level of auxins. The low concentration of ethylene induces the

The physiological role of auxins promote cell growth and cell division, induction of the injured tissue of one to several layers of cells recovering the ability to divide to form Ornamental plants are used especially as decorative houseplants and for landscaping. Ornamental plants are unique for their sheer beauty and variety of leaves. For example Begonias, which have a medicinal value. It is a temperate plant, which is commercially used as a flowering pot plant. It does not produced seeds. It is a winter flowering plant and was developed from cross between other species of the same family. Begonia plant normally has thick, shiny, dark green leaves. Although Begonias can be readily vegetative propagated, they are susceptible to many pathogenic bacteria, fungi, and nematodes [5]. Nowadays, the seaweed market has grown as predicted with prospects to go even further. Therefore potential improvements introduced through the application on *in vitro* techniques are expected to be even higher.

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 259

To initiate cultures, various explants of African violet (*Saintpaulia ionantha* H. Wendl) were excised from 2-month-old intact plants which were grown in greenhouse. The flower buds (3-5 mm), leaf (10 x 10 mm), petiole (10 mm) and peduncle (10 mm) were used as explants for *in vitro* studies [9]. In some cases aseptic seedlings need to be used. Seeds of *Dianthus caryophyllus* were washed with chlorox concentration of 70%, 50%, 30%, 20% and 10% (v/v). First, the seeds were shaken in 70% chlorox with three drops of tween-20 for 15 minutes. They were then washed three times with sterile distilled water. These steps were repeated with other concentrations of chlorox. The seeds were cultured into test tubes containing MS media without hormone under aseptic condition. The work has to be done under sterile conditions. The seeds were germinated in a culture room at the temperature of 25±1 °C with 16 h light and 8 h dark. The growth was evaluated after 30 days. For tissue culture studies of *Gerbera jamesonii* Bolus ex. Hook f. various explants were obtained from 8-week-old aseptic seedlings. *Gerbera* seeds were first soaked in distilled water for 30 min with addition of 1-2 drops of Tween-20, followed by 40% (v/v) Sodium hypochlorite solution and gently agitated. The seeds were then rinsed 3 times with distilled water and then soaked in 70% (v/v) alcohol for 1 min. Finally the seeds were rinsed 3 times with sterile distilled water. Sterilized seeds were cultured in MS basal medium [10]. Surface sterilization process for seeds of cauliflower (*Brassica oleracea* var. *botrytis*) was slightly different; the seeds were soaked in distilled water with 1 or 2 drops of Tween-20 for 20 min, followed by 60% (v/v) Sodium hypochlorite solution, gently agitated for 15 min. The seeds were then rinsed 3 times in distilled water, soaked in 70% ethanol (v/v) for 30 sec and rinsed again in 3 changes of sterile distilled water prior to culturing in MS basal medium [11]. The conventional methods of propagation are problematic due to rapid occurrence of diseases. The production of large numbers of genetically homogenous plants is also very difficult. Plant cell culture technique is an alternative method for mass cloning of Begonia plants and also to overcome the problems occurring in the conventional propagation. The regeneration frequency and average number of shoots per explant varied among the cultivars. Shoot tip size also plays an important role in shoot regeneration efficiency [7]. Wang and Ma reported that shoot tip between 0.2 and 0.5 mm and shoot meristems between 0.1 and 0.2 mm diameter produced only a single shoot [12]. The techniques of stimulating axillary branching or culturing nodal sections *in vitro* are probably most commonly used in micropropagation [13]. Size of the meristem (both shoot tip and nodal explants) of *Floribunda* and miniature roses had significant effect on shoot multiplication; on an average 2.5-5.0 shoots were obtained per culture cycle, dependent on cultivars [14]. Recently, Teixeira de Silva and Fukai published a detailed review on tissue culture of chrysanthemum, which highlights

organogenesis, thin cell layer, and somatic embryogenesis for plant regeneration [15].

MS basal medium [16] was used for these experiments. The constituent of the media was adjusted to 1 liter after the sucrose addition and the pH was adjusted to 5.7 prior to the adding of 7.0 g agar and 0.1 g charcoal to the media. Media was autoclaved at 121 C 1.5 kpa

*5.1.2. Media and plant growth regulators roles* 

*5.1.1. Explants sterilization procedure* 

Modern techniques of propagation through tissue culture technique have been developed to meet the demand of the horticultural industries including nursery industries. For pot plant production, the priority is to obtain early, synchronized and profuse flowering, together with a compact and homogenous plant size, rather than continuous flowering. For ornamental pot plant production to be successful, an efficient method for flower induction in small plantlets is thus required. Thus, the application of plant tissue culture technique is always required. The technology is widely applied in both research and development of improved crops [6]. Rout *et al*. reported that about 156 ornamental genera were propagated through tissue culture in different commercial laboratories worldwide [7].

## **5. Method used**

#### **5.1. Surface sterilization**

Surface of plant parts carry a wide range of microbial contaminants. The presence of any contaminant will interfere with the growth of explant or cultures and fungal or bacterial explant contamination in plant cultures is usually detectable 1-14 days after culturing. Therefore, sterilization or disinfection of tissues is necessary in order to eradicate surface microorganism. In order to disinfect plant tissues, 5-50% (v/v) commercial bleach Clorox (Sodium hypochlorite), 70% (v/v) alcohol and a few drops of Tween 20 can be used in sterilization technique. Pierik *et al.* had suggested that sterilization plant for a few seconds in alcohol is not sufficient to kill all microorganisms and after this they are usually treated with sodium hypochlorite [2]. Diluted solution of sodium hypochlorite (0.25-2.63%) (v/v) is used as a disinfectant and tween 20 is an emulsifier which is added at the rate of 1 drop per 100 ml of solution. The development of techniques for the culture of isolated plant organs, tissues and cells have led to several exciting opportunities in the area of plant biotechnology, and allowed widespread use of cell culture for *in vitro* genetic manipulation, plant propagation and production of commercially useful products. The techniques of cultivating cells and tissues have been referred to sometimes as "aseptic culture of plants". Therefore, the absence of contaminants is assumed to be a fundamental requisite *in vitro*. Surface sterilization of ornamental plants is difficult as they lack a thick protective surface, and therefore sodium hypochlorite and similar agents can easily damage the delicate tissues. The scope of these techniques has been extended for use in bioprocess technology for production of high value chemicals of immense commercial value in the pharmaceutical and nutraceutical sectors [8]. Finally, the instruments including forceps and scalpels were sterilized by dipping them into hot bead sterilizer at 250ºC and allowed to cool. Glassware, empty test tubes, empty flasks, petri dishes, filter paper and distilled water can be sterilized using an autoclave at 121ºC, 105 kPa, for 20 minutes. The bottles and glassware should not be too tightly packed and their tops should be loosen during autoclaving.

#### *5.1.1. Explants sterilization procedure*

258 Plant Science

expected to be even higher.

**5. Method used** 

**5.1. Surface sterilization** 

tops should be loosen during autoclaving.

potential improvements introduced through the application on *in vitro* techniques are

Modern techniques of propagation through tissue culture technique have been developed to meet the demand of the horticultural industries including nursery industries. For pot plant production, the priority is to obtain early, synchronized and profuse flowering, together with a compact and homogenous plant size, rather than continuous flowering. For ornamental pot plant production to be successful, an efficient method for flower induction in small plantlets is thus required. Thus, the application of plant tissue culture technique is always required. The technology is widely applied in both research and development of improved crops [6]. Rout *et al*. reported that about 156 ornamental genera were propagated

Surface of plant parts carry a wide range of microbial contaminants. The presence of any contaminant will interfere with the growth of explant or cultures and fungal or bacterial explant contamination in plant cultures is usually detectable 1-14 days after culturing. Therefore, sterilization or disinfection of tissues is necessary in order to eradicate surface microorganism. In order to disinfect plant tissues, 5-50% (v/v) commercial bleach Clorox (Sodium hypochlorite), 70% (v/v) alcohol and a few drops of Tween 20 can be used in sterilization technique. Pierik *et al.* had suggested that sterilization plant for a few seconds in alcohol is not sufficient to kill all microorganisms and after this they are usually treated with sodium hypochlorite [2]. Diluted solution of sodium hypochlorite (0.25-2.63%) (v/v) is used as a disinfectant and tween 20 is an emulsifier which is added at the rate of 1 drop per 100 ml of solution. The development of techniques for the culture of isolated plant organs, tissues and cells have led to several exciting opportunities in the area of plant biotechnology, and allowed widespread use of cell culture for *in vitro* genetic manipulation, plant propagation and production of commercially useful products. The techniques of cultivating cells and tissues have been referred to sometimes as "aseptic culture of plants". Therefore, the absence of contaminants is assumed to be a fundamental requisite *in vitro*. Surface sterilization of ornamental plants is difficult as they lack a thick protective surface, and therefore sodium hypochlorite and similar agents can easily damage the delicate tissues. The scope of these techniques has been extended for use in bioprocess technology for production of high value chemicals of immense commercial value in the pharmaceutical and nutraceutical sectors [8]. Finally, the instruments including forceps and scalpels were sterilized by dipping them into hot bead sterilizer at 250ºC and allowed to cool. Glassware, empty test tubes, empty flasks, petri dishes, filter paper and distilled water can be sterilized using an autoclave at 121ºC, 105 kPa, for 20 minutes. The bottles and glassware should not be too tightly packed and their

through tissue culture in different commercial laboratories worldwide [7].

To initiate cultures, various explants of African violet (*Saintpaulia ionantha* H. Wendl) were excised from 2-month-old intact plants which were grown in greenhouse. The flower buds (3-5 mm), leaf (10 x 10 mm), petiole (10 mm) and peduncle (10 mm) were used as explants for *in vitro* studies [9]. In some cases aseptic seedlings need to be used. Seeds of *Dianthus caryophyllus* were washed with chlorox concentration of 70%, 50%, 30%, 20% and 10% (v/v). First, the seeds were shaken in 70% chlorox with three drops of tween-20 for 15 minutes. They were then washed three times with sterile distilled water. These steps were repeated with other concentrations of chlorox. The seeds were cultured into test tubes containing MS media without hormone under aseptic condition. The work has to be done under sterile conditions. The seeds were germinated in a culture room at the temperature of 25±1 °C with 16 h light and 8 h dark. The growth was evaluated after 30 days. For tissue culture studies of *Gerbera jamesonii* Bolus ex. Hook f. various explants were obtained from 8-week-old aseptic seedlings. *Gerbera* seeds were first soaked in distilled water for 30 min with addition of 1-2 drops of Tween-20, followed by 40% (v/v) Sodium hypochlorite solution and gently agitated. The seeds were then rinsed 3 times with distilled water and then soaked in 70% (v/v) alcohol for 1 min. Finally the seeds were rinsed 3 times with sterile distilled water. Sterilized seeds were cultured in MS basal medium [10]. Surface sterilization process for seeds of cauliflower (*Brassica oleracea* var. *botrytis*) was slightly different; the seeds were soaked in distilled water with 1 or 2 drops of Tween-20 for 20 min, followed by 60% (v/v) Sodium hypochlorite solution, gently agitated for 15 min. The seeds were then rinsed 3 times in distilled water, soaked in 70% ethanol (v/v) for 30 sec and rinsed again in 3 changes of sterile distilled water prior to culturing in MS basal medium [11]. The conventional methods of propagation are problematic due to rapid occurrence of diseases. The production of large numbers of genetically homogenous plants is also very difficult. Plant cell culture technique is an alternative method for mass cloning of Begonia plants and also to overcome the problems occurring in the conventional propagation. The regeneration frequency and average number of shoots per explant varied among the cultivars. Shoot tip size also plays an important role in shoot regeneration efficiency [7]. Wang and Ma reported that shoot tip between 0.2 and 0.5 mm and shoot meristems between 0.1 and 0.2 mm diameter produced only a single shoot [12]. The techniques of stimulating axillary branching or culturing nodal sections *in vitro* are probably most commonly used in micropropagation [13]. Size of the meristem (both shoot tip and nodal explants) of *Floribunda* and miniature roses had significant effect on shoot multiplication; on an average 2.5-5.0 shoots were obtained per culture cycle, dependent on cultivars [14]. Recently, Teixeira de Silva and Fukai published a detailed review on tissue culture of chrysanthemum, which highlights organogenesis, thin cell layer, and somatic embryogenesis for plant regeneration [15].

#### *5.1.2. Media and plant growth regulators roles*

MS basal medium [16] was used for these experiments. The constituent of the media was adjusted to 1 liter after the sucrose addition and the pH was adjusted to 5.7 prior to the adding of 7.0 g agar and 0.1 g charcoal to the media. Media was autoclaved at 121 C 1.5 kpa for 20 minutes. Media were then dispensed into sterile plastic vials containing 20-25 ml of aliquots, inside the laminar air flow cabinet. For culturing of *Pereskia grandifolia* the axillary bud explants were sliced and cultured on MS medium consisted of 0.1-10 mg/l BAP and 30 mg/l adenine as well as BAP and NAA. Petioles obtained from aseptically grown young plantlet of *Gerbera jasmesonii* were used as source of explants. Leaves and petioles were cultured for shoot induction on MS media containing BAP and NAA at various concentrations. Plant regeneration from leaf disk callus of *Begonia elatior* was achieved on MS medium supplemented with 1.0 mg/l Kn and 0.1 mg/l zeatin [17]. Liquid medium seems to be more effective for shoot regeneration and root induction, which is due to better aeration. Simmonds and Werry used liquid medium for enhancing the micropropagation profile of *Begonia hiemalis* [18]. Liquid media have been used for plant cells, somatic embryos and cell suspension cultures in either agitated flasks or various types of bioreactors [19-24]. Wated et al. compared performance of agar-solidified medium and interfacial membrane drafts floating on liquid medium for shoot multiplication and root induction [25]. The regenerated shoots were rooted on half-strength MS medium supplemented with 0.1 mg/l NAA and 0.2 mg/l Kn. Nearly 300 plantlets of each cultivar were transferred to soil with 95% survival rate [26].

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 261

al. [37], who developed a large scale propagation system of *Cyclamen* from embryogenic cell suspension cultures. Kumari *et al*. developed an efficient protocol for micropropagation of *Chrysanthemum* on MS medium supplemented with 1.0-2.5 μM TDZ [38]. Castillo and Smith induced direct somatic embryogenesis from petiole and leaf blade explants of *Begonia gracilis*  on MS medium supplemented with 0.5 mg/l kinetin and 2% (v/v) coconut water [5]. Kim et al. established a large-scale propagation of *chrysanthemum* through bioreactor system, and

*In vitro* propagation through meristem culture is the best possible means of virus elimination and produces a large numbers of plants in a short span of time. It is a powerful tool for large-scale propagation of ornamental plants. The term 'meristem culture' specially means that a meristem with no leaf primordial or at most 1-2 leaf primordial which are excised and cultured. The pathway of regeneration undergoes several steps. Starting with an isolated explant, with de-differentiation followed by re-differentiation and organization into meristematic centers. More than 600 million micropropagated plants are produced every year in the world [40]. Micropropagation is one of the few areas of plant tissue culture in which the techniques have been applied commercially. To circumvent these impediments, clonal or vegetative propagation has been deployed for recovering dominant, additive and epistatic genetic effects to select superior genotypes. Plant tissue culture methods offer an important option for effective multiplication and improvement of ornamental plants within

*Gerbera jamesonii* is an ornamental flowering perennial belonging to the Asteraceae family. This plant is very well known to be planted as cut flowers, bedding plant and also pot crops. The *in vitro* shoots were successfully obtained in petiole explants of *Gerbera jamesonii*  [10]. The adventitious shoots were observed on MS medium supplemented with BA (1.0 mg/l) and NAA (0.5 mg/l). The developed shoots were subcultured every two week interval for shoot elongation. The elongated shoots were transferred for rooting on MS media with IAA (2.0 mg/l). The developed plantlets were maintained in the following acclimatization conditions : the garden soil (black soil: red soil, 2:1) which gave best result with 86.0 ± 0.9% survival rates, followed by vermiculite with 73 ±1.3% survival rates. In this study, plantlets established from *in vitro* regeneration of *Gerbera jamesonii* were morphologically identical to the mother plant and developed normally and produced flowers after 6 months being

Leaf and petiole explants were cultured on MS medium for shoot induction containing BAP (0.5-2.0 mg/l) and NAA (0.5-1.0 mg/l). Normal adventitious shoots of *Gerbera jamesonii* were

obtained 5000 plantlets after 12 weeks of culture in 10±l column type bioreactor [39].

**6. Results** 

**6.1. Micropropagation** 

a limited time frame.

*6.1.1. Gerbera jamesonii* 

transplanted to the greenhouse [41,42].

*6.1.1.1. Multiple shoots induction* 

#### *5.1.3. Active chemical roles*

During *in vitro* conditions, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. When explants are first placed onto a nutrient medium, there is often an initial leakage of ions from damaged cells, especially metallic cations (Na+, Ca2+, K+, Mg2+) for the 1-2 days, so that the concentration in the plant tissues actually decreases [27]. Cells then commence active absorption and the internal concentration slowly rises. Phosphate and nitrogen (particularly ammonium) are absorbed more rapidly than other ions. Both growth and morphogenesis in tissues cultures are markedly influenced by the availability of nitrogen and the form in which it is presented [28]. Chemical and substances are synthesized in particular cells and are transferred to other cells, which in extremely small quantities influence the development process. The plant growth regulators are implicated in many biological processes in ornamental plants, including cell division, root and floral initiation, fruit development, senescence and abiotic stress responses.

The rooting efficiency enhanced by addition of 0.05% Poly vinyl pyrrolidone (PVP) in the culture medium containing 0.5 mg/l IBA [29]. The addition of PVP helps in oxidizing polyphenols leached in the medium, and promotes high rate of organogenesis. Dijkshoorn-Dekker studied the influence of light and temperature on propagation profile of *Ficus benjamina* [30]*.* Propagation of different *Ficus* species by using shoot tips or axillary bud explants had been reported [29,31-34]. In the most cases, shoots were rooted in hormone free medium. Both orientation of the petiole explants and auxin transport system are crucial factors for the induction of somatic embryogenesis of *Saintpaulia* [35], and TDZ helped in the development of somatic embryos. Winkelmann *et al*. used cell suspension culture of *Cyclamen* for rapid development of somatic embryos [36], and later on followed by Hohe et al. [37], who developed a large scale propagation system of *Cyclamen* from embryogenic cell suspension cultures. Kumari *et al*. developed an efficient protocol for micropropagation of *Chrysanthemum* on MS medium supplemented with 1.0-2.5 μM TDZ [38]. Castillo and Smith induced direct somatic embryogenesis from petiole and leaf blade explants of *Begonia gracilis*  on MS medium supplemented with 0.5 mg/l kinetin and 2% (v/v) coconut water [5]. Kim et al. established a large-scale propagation of *chrysanthemum* through bioreactor system, and obtained 5000 plantlets after 12 weeks of culture in 10±l column type bioreactor [39].

### **6. Results**

260 Plant Science

95% survival rate [26].

*5.1.3. Active chemical roles* 

for 20 minutes. Media were then dispensed into sterile plastic vials containing 20-25 ml of aliquots, inside the laminar air flow cabinet. For culturing of *Pereskia grandifolia* the axillary bud explants were sliced and cultured on MS medium consisted of 0.1-10 mg/l BAP and 30 mg/l adenine as well as BAP and NAA. Petioles obtained from aseptically grown young plantlet of *Gerbera jasmesonii* were used as source of explants. Leaves and petioles were cultured for shoot induction on MS media containing BAP and NAA at various concentrations. Plant regeneration from leaf disk callus of *Begonia elatior* was achieved on MS medium supplemented with 1.0 mg/l Kn and 0.1 mg/l zeatin [17]. Liquid medium seems to be more effective for shoot regeneration and root induction, which is due to better aeration. Simmonds and Werry used liquid medium for enhancing the micropropagation profile of *Begonia hiemalis* [18]. Liquid media have been used for plant cells, somatic embryos and cell suspension cultures in either agitated flasks or various types of bioreactors [19-24]. Wated et al. compared performance of agar-solidified medium and interfacial membrane drafts floating on liquid medium for shoot multiplication and root induction [25]. The regenerated shoots were rooted on half-strength MS medium supplemented with 0.1 mg/l NAA and 0.2 mg/l Kn. Nearly 300 plantlets of each cultivar were transferred to soil with

During *in vitro* conditions, plantlets are grown under fixed and controlled environment in sterile formulated medium which contained macronutrients, micronutrients, vitamins and plant growth regulators. When explants are first placed onto a nutrient medium, there is often an initial leakage of ions from damaged cells, especially metallic cations (Na+, Ca2+, K+, Mg2+) for the 1-2 days, so that the concentration in the plant tissues actually decreases [27]. Cells then commence active absorption and the internal concentration slowly rises. Phosphate and nitrogen (particularly ammonium) are absorbed more rapidly than other ions. Both growth and morphogenesis in tissues cultures are markedly influenced by the availability of nitrogen and the form in which it is presented [28]. Chemical and substances are synthesized in particular cells and are transferred to other cells, which in extremely small quantities influence the development process. The plant growth regulators are implicated in many biological processes in ornamental plants, including cell division, root

and floral initiation, fruit development, senescence and abiotic stress responses.

The rooting efficiency enhanced by addition of 0.05% Poly vinyl pyrrolidone (PVP) in the culture medium containing 0.5 mg/l IBA [29]. The addition of PVP helps in oxidizing polyphenols leached in the medium, and promotes high rate of organogenesis. Dijkshoorn-Dekker studied the influence of light and temperature on propagation profile of *Ficus benjamina* [30]*.* Propagation of different *Ficus* species by using shoot tips or axillary bud explants had been reported [29,31-34]. In the most cases, shoots were rooted in hormone free medium. Both orientation of the petiole explants and auxin transport system are crucial factors for the induction of somatic embryogenesis of *Saintpaulia* [35], and TDZ helped in the development of somatic embryos. Winkelmann *et al*. used cell suspension culture of *Cyclamen* for rapid development of somatic embryos [36], and later on followed by Hohe et

#### **6.1. Micropropagation**

*In vitro* propagation through meristem culture is the best possible means of virus elimination and produces a large numbers of plants in a short span of time. It is a powerful tool for large-scale propagation of ornamental plants. The term 'meristem culture' specially means that a meristem with no leaf primordial or at most 1-2 leaf primordial which are excised and cultured. The pathway of regeneration undergoes several steps. Starting with an isolated explant, with de-differentiation followed by re-differentiation and organization into meristematic centers. More than 600 million micropropagated plants are produced every year in the world [40]. Micropropagation is one of the few areas of plant tissue culture in which the techniques have been applied commercially. To circumvent these impediments, clonal or vegetative propagation has been deployed for recovering dominant, additive and epistatic genetic effects to select superior genotypes. Plant tissue culture methods offer an important option for effective multiplication and improvement of ornamental plants within a limited time frame.

#### *6.1.1. Gerbera jamesonii*

*Gerbera jamesonii* is an ornamental flowering perennial belonging to the Asteraceae family. This plant is very well known to be planted as cut flowers, bedding plant and also pot crops. The *in vitro* shoots were successfully obtained in petiole explants of *Gerbera jamesonii*  [10]. The adventitious shoots were observed on MS medium supplemented with BA (1.0 mg/l) and NAA (0.5 mg/l). The developed shoots were subcultured every two week interval for shoot elongation. The elongated shoots were transferred for rooting on MS media with IAA (2.0 mg/l). The developed plantlets were maintained in the following acclimatization conditions : the garden soil (black soil: red soil, 2:1) which gave best result with 86.0 ± 0.9% survival rates, followed by vermiculite with 73 ±1.3% survival rates. In this study, plantlets established from *in vitro* regeneration of *Gerbera jamesonii* were morphologically identical to the mother plant and developed normally and produced flowers after 6 months being transplanted to the greenhouse [41,42].

#### *6.1.1.1. Multiple shoots induction*

Leaf and petiole explants were cultured on MS medium for shoot induction containing BAP (0.5-2.0 mg/l) and NAA (0.5-1.0 mg/l). Normal adventitious shoots of *Gerbera jamesonii* were

successively obtained from petiole explants cultured on MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) with 94.3% regeneration rate and 9.3 shoots per explant, followed by BAP (1.5 mg/l) and NAA (1.0 mg/l) with 83.1% regeneration rates and 8.3 shoots per explant. Pierik et al. stated that the addition of strong auxin such as NAA in combination with BAP promoted shoots induction in plant tissue culture [2]. In this study, higher concentration of auxin, NAA (2.0 mg/l) in combination with lower concentration of BAP (1.0 mg/l) showed the lowest shoots regeneration rates (4.6) with the lowest number of shoots (1.6). Son et al. investigated the micropropagation of different plant varieties using the plant buds as explants [43]. They found that the best hormone combination for the *in vitro* initiation of *Gerbera jamesonii* shoots was 3 mg/l BAP + 0.1 mg/l IAA producing 11.29 number of shoots per explant on MS medium.

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 263

the origin of explants used for the establishment of callus culture, the cellular/tissue differentiation status, external plant growth regulators, culture media and culture conditions [46]. Cellular competence to plant hormones is understood as the status in which a cell must possess the ability to perceive a transducer and respond to a signal [47]. Organogenesis refers to the formation of shoots/roots. The callus may remain in a differentiated condition regardless of the hormones and nutrients to which it is exposed the secondary metabolites and these metabolites have biological activity [48-50]. Organ formation generally follows cessation of unlimited proliferation of callus. Individual cells or group of cells of smaller dimensions may form small nets of cells scattered throughout the callus tissue, the so-called meristemoids. These meristemoids become transformed into cyclic nodules from which shoot bud or root primordia may grow as shoots/roots. Shoot bud formation may decrease with age and subculture duration of the callus tissue but the capacity of rooting may persist for longer period. In some calli, rooting occurs more often than in other forms of organogenesis. During organogenesis, if the roots are first formed, then it is very difficult to induce adventitious shoot bud formation from the same callus tissue. If the shoots are first formed, it may form roots later on or may remain in rootless condition unless and until the shoots are transformed to another medium or hormone less medium or conditions that induce root formation. In certain cases root and shoot formation may occur simultaneously, but organ connection (vascular connection) between root and shoot primordial is essential for the regeneration of complete plantlet from the culture. Shoot formation followed by rooting is the general feature of organogenesis. The color of callus tissue may remain unchanged during rhizogenesis or may develop yellow pigmentation. During shoot bud formation, the callus tissue generally develops green or pale green pigmentation. Organogenesis is a process by which a cell or group of cells differentiate to form organs. It is the reflection of the intrinsic genetic constitution of a taxon [51]. Since recovery of plants is the usual objective, regeneration of shoots is of greater interest. Organogenesis is commonly induced by manipulation of exogenous phytohormone

levels and occurs either directly from explant tissue or through callus.

*Saintpaulia ionantha* known worldwide as African violet belongs to the family Gesneriaceae. African violet is one of the most popular ornamental plants. The complete plant regeneration was obtained from leaf, petiole, peduncle and floral parts of the plant. For shoot regeneration MS medium supplemented with IAA (1.0 mg/l) with Zeatin (2.0 mg/l). The developed plantlets were transferred for acclimatization, whereby *in vitro* plantlets were transferred into planting pots containing mixed soil, which is a mixture of compost, sand and black soil with the ratio of 1:1:2. The morphological characters were compared to the

The commercial exploitation of the ornamental plants for the production and conventional propagation is hampered due to their poor seed viability, low rate of germination and poor rooting ability of the vegetative cuttings. Somatic embryogenesis is an alternative method.

*6.2.1. Saintpaulia ionantha* 

mother plants [52].

*6.2.2. Somatic embryogenesis* 

#### *6.1.1.2. Rooting and hardening*

The induced shoots were best rooted on MS media supplemented with BAP (0.1 mg/l) with frequency of 73.7% and 22.1 roots per explant, while Son et. al.[43] found that MS medium supplemented with 2.0 mg/l NAA was the best medium for *in vitro* rooting of the shoots (94.0%). The developed plantlets were maintained in the following acclimatization conditions: the garden soil (black soil: red soil, 2:1) which gave the best results with 86.0 % survival rates, followed by vermiculite with 73% survival rates. The regenerated plantlets failed to survive, when they were cultured in the autoclaved garden soil (black soil: red soil, 2:1). The plantlets established from *in vitro* regeneration of *Gerbera jamesonii* were morphologically identical to the mother plant and developed normally and also produced flowers after 6 months being transplanted to the greenhouse.

#### *6.1.2. Pereskia grandifolia Haworth var. grandifolia*

A protocol for an *in vitro* propagation was developed for the ornamental plant *Pereskia grandifolia Haworth var. grandifolia* from axillary bud explants. Optimum multiplication of shoots was achieved on MS [16] medium supplemented with 3.0 mg/l BAP and 30.0 mg/l adenine. Plants were maintained *in vitro* on MS medium while callus were induced on MS basal medium supplemented with the combination of 5.0 mg/l BAP and 5.0 mg/l NAA. The somatic embryogenic callus of the plant species was induced by Chuah and Chan on B5 medium supplemented with 6.0 mg/l 2, 4-D [44,45].

#### **6.2. Organogenesis**

Regeneration in plant tissue culture will be successful by maintaining various factors involved, including media factors and environmental factors. The media factors include media constituents, macronutrients, micronutrients, vitamins, amino acids, carbon source, complex nutritive mixtures, gelling agents, activated charcoal, plant growth regulators and pH of the medium. Environmental factors on the other hand are the culture conditions under which explants are maintained. The environmental factors involved include the temperature and illumination of the culture room, agitation process and incubation period of the cultures [4]. For the initiation of callus culture, the following factors are important-: the origin of explants used for the establishment of callus culture, the cellular/tissue differentiation status, external plant growth regulators, culture media and culture conditions [46]. Cellular competence to plant hormones is understood as the status in which a cell must possess the ability to perceive a transducer and respond to a signal [47]. Organogenesis refers to the formation of shoots/roots. The callus may remain in a differentiated condition regardless of the hormones and nutrients to which it is exposed the secondary metabolites and these metabolites have biological activity [48-50]. Organ formation generally follows cessation of unlimited proliferation of callus. Individual cells or group of cells of smaller dimensions may form small nets of cells scattered throughout the callus tissue, the so-called meristemoids. These meristemoids become transformed into cyclic nodules from which shoot bud or root primordia may grow as shoots/roots. Shoot bud formation may decrease with age and subculture duration of the callus tissue but the capacity of rooting may persist for longer period. In some calli, rooting occurs more often than in other forms of organogenesis. During organogenesis, if the roots are first formed, then it is very difficult to induce adventitious shoot bud formation from the same callus tissue. If the shoots are first formed, it may form roots later on or may remain in rootless condition unless and until the shoots are transformed to another medium or hormone less medium or conditions that induce root formation. In certain cases root and shoot formation may occur simultaneously, but organ connection (vascular connection) between root and shoot primordial is essential for the regeneration of complete plantlet from the culture. Shoot formation followed by rooting is the general feature of organogenesis. The color of callus tissue may remain unchanged during rhizogenesis or may develop yellow pigmentation. During shoot bud formation, the callus tissue generally develops green or pale green pigmentation. Organogenesis is a process by which a cell or group of cells differentiate to form organs. It is the reflection of the intrinsic genetic constitution of a taxon [51]. Since recovery of plants is the usual objective, regeneration of shoots is of greater interest. Organogenesis is commonly induced by manipulation of exogenous phytohormone levels and occurs either directly from explant tissue or through callus.

#### *6.2.1. Saintpaulia ionantha*

262 Plant Science

number of shoots per explant on MS medium.

flowers after 6 months being transplanted to the greenhouse.

*6.1.2. Pereskia grandifolia Haworth var. grandifolia* 

medium supplemented with 6.0 mg/l 2, 4-D [44,45].

**6.2. Organogenesis** 

*6.1.1.2. Rooting and hardening* 

successively obtained from petiole explants cultured on MS medium supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) with 94.3% regeneration rate and 9.3 shoots per explant, followed by BAP (1.5 mg/l) and NAA (1.0 mg/l) with 83.1% regeneration rates and 8.3 shoots per explant. Pierik et al. stated that the addition of strong auxin such as NAA in combination with BAP promoted shoots induction in plant tissue culture [2]. In this study, higher concentration of auxin, NAA (2.0 mg/l) in combination with lower concentration of BAP (1.0 mg/l) showed the lowest shoots regeneration rates (4.6) with the lowest number of shoots (1.6). Son et al. investigated the micropropagation of different plant varieties using the plant buds as explants [43]. They found that the best hormone combination for the *in vitro* initiation of *Gerbera jamesonii* shoots was 3 mg/l BAP + 0.1 mg/l IAA producing 11.29

The induced shoots were best rooted on MS media supplemented with BAP (0.1 mg/l) with frequency of 73.7% and 22.1 roots per explant, while Son et. al.[43] found that MS medium supplemented with 2.0 mg/l NAA was the best medium for *in vitro* rooting of the shoots (94.0%). The developed plantlets were maintained in the following acclimatization conditions: the garden soil (black soil: red soil, 2:1) which gave the best results with 86.0 % survival rates, followed by vermiculite with 73% survival rates. The regenerated plantlets failed to survive, when they were cultured in the autoclaved garden soil (black soil: red soil, 2:1). The plantlets established from *in vitro* regeneration of *Gerbera jamesonii* were morphologically identical to the mother plant and developed normally and also produced

A protocol for an *in vitro* propagation was developed for the ornamental plant *Pereskia grandifolia Haworth var. grandifolia* from axillary bud explants. Optimum multiplication of shoots was achieved on MS [16] medium supplemented with 3.0 mg/l BAP and 30.0 mg/l adenine. Plants were maintained *in vitro* on MS medium while callus were induced on MS basal medium supplemented with the combination of 5.0 mg/l BAP and 5.0 mg/l NAA. The somatic embryogenic callus of the plant species was induced by Chuah and Chan on B5

Regeneration in plant tissue culture will be successful by maintaining various factors involved, including media factors and environmental factors. The media factors include media constituents, macronutrients, micronutrients, vitamins, amino acids, carbon source, complex nutritive mixtures, gelling agents, activated charcoal, plant growth regulators and pH of the medium. Environmental factors on the other hand are the culture conditions under which explants are maintained. The environmental factors involved include the temperature and illumination of the culture room, agitation process and incubation period of the cultures [4]. For the initiation of callus culture, the following factors are important-: *Saintpaulia ionantha* known worldwide as African violet belongs to the family Gesneriaceae. African violet is one of the most popular ornamental plants. The complete plant regeneration was obtained from leaf, petiole, peduncle and floral parts of the plant. For shoot regeneration MS medium supplemented with IAA (1.0 mg/l) with Zeatin (2.0 mg/l). The developed plantlets were transferred for acclimatization, whereby *in vitro* plantlets were transferred into planting pots containing mixed soil, which is a mixture of compost, sand and black soil with the ratio of 1:1:2. The morphological characters were compared to the mother plants [52].

#### *6.2.2. Somatic embryogenesis*

The commercial exploitation of the ornamental plants for the production and conventional propagation is hampered due to their poor seed viability, low rate of germination and poor rooting ability of the vegetative cuttings. Somatic embryogenesis is an alternative method. However, there is lack of information for the embryo induction process [53]. The aim or work was to study the germination capability and development of somatic embryos (SEs) from ornamental plants. A sustainable plant regeneration system *in vitro* through somatic embryos from mature sexual embryos has been reported in *Clitoria ternatea*. Somatic embryos developed through callus from seedling roots on hormone-free MS medium (MS1). Addition of growth hormones, Kn 0.5 mg/l (MS2) or Kn +IAA 0.5 mg/l of each (MS3) induced direct somatic embryos, in high frequency, on split root and hypocotyl systems. The embryogenic potential varied with the organ, roots or hypocotyls, and also with the medium. The morphogenetic capacity of the somatic embryos is retained for more than 2 years by sub-culturing at intervals of 4 weeks on MS3 in complete darkness. Somatic embryos, under the appropriate subculture conditions (16 h light/8 h dark photoperiod at 24± 1 °C on media MS3, MS4 and MS5), resulted in recurrent-somatic embryogenesis and was profuse at the shoot and root apices of the somatic embryos. Mature somatic embryos were transplanted to MS1 to stimulate germination and plantlet regeneration. Plantlets, developed from primary and secondary embryos on MS1 were successfully hardened and grown in natural outdoor conditions. The morphology and histology of the somatic embryo and plantlet and the culture conditions for continuous production of plantlets through direct somatic embryogenesis are discussed. In our laboratory, plant regeneration of *Clitoria ternatea* was reported from leaf explants cultured on DKW (Driver and Kuniyuki) medium supplemented with various concentrations of NAA and BAP [54].

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 265

The *in vitro* regeneration of the carnation plant species was established from aseptic shoots produced from surface sterilized seeds grown in basal MS medium. High percentage of grown seeds was obtained (50-90%) after 3 weeks of culturing the seeds. All the plantlets were rooted efficiently on the multiplication MS medium without hormone supplementation. This may be due to the presence of endogenous auxins which causes rooting as it was stated by Mosleh et al. [56]. After 4 weeks, full grown plantlets were transferred to sterile soil at ratio of 1:1:1 for garden soil, sand and loam for acclimatization process. They were maintained in the culture room at 25±1°C with 16 hours light and 8 hours dark planted in pots and covered with polystyrene plastic bags. The growth was encouraging and after 4 weeks in the culture room, well grown and healthy plantlets were transferred to the greenhouse. Survival rate of the acclimatized plantlets was 78% and it showed that the most of the plantlets successfully survived after transferred to the greenhouse. Best response for hardening of the plant species was also obtained by Aamir et al. [57] (95%) in mixture containing sand, peat and soil (1:1:1) under natural light conditions

The complete plant regeneration was obtained from leaf, petiole, peduncle and floral parts of the plant. For shoot regeneration, MS medium supplemented with IAA (1.0 mg/l) and Zeatin (2.0 mg/l) induced the highest number of shoots (15.0) in 8 weeks. While shoots regeneration and multiplication obtained from the same plant by Azura et al. [59] with maximum induction rate on MS medium supplemented with 3.0 mg/l BAP and 1.0 mg/l of NAA. It was reported by Hasbullah et al. [10] that a good combination of cytokinins and auxin in the plant culture medium enhanced good shoot formation and plantlet regeneration. Most of the developed plantlets were hardened (84%) and acclimatized in the greenhouse by transferring the plantlets into planting pots containing mixed soil, which is a mixture of compost, sand and black soil with the ratio of 1:1:2. Meanwhile, Khan et al. found that among the different potting mixes used for the acclimatization of rooted plantlets, 100%

However, the obtained new plants failed to flower even after twelve months from planting. The morphological characters of these plantlets were compared to those of the mother plants. They were found to be different in some of their morphological characters such as plant height, leaf size and leaf texture and showed similarity in leaf arrangement and leaf margin and they showed unstable morphological characters. But variations in flowering period, number of flowers per plant and flower morphology were observed by Jain [17] in the plants directly regenerated from leaf disk explants. So, he concluded that the cytokinins, benzylaminopurine and zeatin tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. An attempt to induce *in vitro* flowering from african violet was also reported by Daud and Taha [9]. They found that the floral buds were formed *in vitro* with sepal (calyx) and petal (corolla) but did

not show any formation of reproductive organs (stamens or pistils).

*6.2.3. Dianthus caryophyllus* 

*6.2.4. Saintpaulia ionantha* 

sand was found to be the best [59].

[58].

Embryogenic callus was induced from leaf explants of *Gerbera jamesonii* Bolus ex. Hook f. in cell suspension cultures. A cream friable embryogenic callus was formed within two weeks when leaf explants were cultured on MS medium containing 2,4-D. hormone (1.0 to 2.0 mg/l). A hundred percent (100%) induction frequency was obtained in 2,4-D concentration range of 1.7-2.0 mg/l. While maximum percentage response for somatic embryos induction (64.56%) from callus clumps was obtained on MS medium fortified with BAP (0.5 mg/1) and NAA (1.0 mg/1) by Ranjan and Gaurav [27]. For proliferation, embryogenic callus was transferred to MS liquid medium containing the same hormone; 2,4-D with a small amount of NAA and subcultured at 2 weeks interval. Induction of somatic embryos different stages (globular, heart and torpedo) were observed after 2 weeks of culture. Somatic embryos were developed in MS suspension medium containing 1.0 to 2.0 mg/l 2,4-D with 0.1 or 1.0 mg/l NAA and the globular embryos were further differentiated into the cotyledonary phase embryos. The addition of 5.0 mg/l amino acids (L-glutamine or L-proline) to the culture media, in the range of the tested medium condition, yielded higher enhancement of the embryo growth and development. Transfering of individual embryos onto a fresh basal MS medium without plant growth regulators enabled the achievement of complete maturation. Relatively, only a small number of the induced embryos developed shoots and roots when they were transferred to MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA in addition of 3% (w/v) sucrose and 0.8% (w/v) agar. Nearly, 11% of somatic embryos were able to be converted to fertile plants. This similar result was supported by other authors [3,55].

#### *6.2.3. Dianthus caryophyllus*

264 Plant Science

[54].

[3,55].

However, there is lack of information for the embryo induction process [53]. The aim or work was to study the germination capability and development of somatic embryos (SEs) from ornamental plants. A sustainable plant regeneration system *in vitro* through somatic embryos from mature sexual embryos has been reported in *Clitoria ternatea*. Somatic embryos developed through callus from seedling roots on hormone-free MS medium (MS1). Addition of growth hormones, Kn 0.5 mg/l (MS2) or Kn +IAA 0.5 mg/l of each (MS3) induced direct somatic embryos, in high frequency, on split root and hypocotyl systems. The embryogenic potential varied with the organ, roots or hypocotyls, and also with the medium. The morphogenetic capacity of the somatic embryos is retained for more than 2 years by sub-culturing at intervals of 4 weeks on MS3 in complete darkness. Somatic embryos, under the appropriate subculture conditions (16 h light/8 h dark photoperiod at 24± 1 °C on media MS3, MS4 and MS5), resulted in recurrent-somatic embryogenesis and was profuse at the shoot and root apices of the somatic embryos. Mature somatic embryos were transplanted to MS1 to stimulate germination and plantlet regeneration. Plantlets, developed from primary and secondary embryos on MS1 were successfully hardened and grown in natural outdoor conditions. The morphology and histology of the somatic embryo and plantlet and the culture conditions for continuous production of plantlets through direct somatic embryogenesis are discussed. In our laboratory, plant regeneration of *Clitoria ternatea* was reported from leaf explants cultured on DKW (Driver and Kuniyuki) medium supplemented with various concentrations of NAA and BAP

Embryogenic callus was induced from leaf explants of *Gerbera jamesonii* Bolus ex. Hook f. in cell suspension cultures. A cream friable embryogenic callus was formed within two weeks when leaf explants were cultured on MS medium containing 2,4-D. hormone (1.0 to 2.0 mg/l). A hundred percent (100%) induction frequency was obtained in 2,4-D concentration range of 1.7-2.0 mg/l. While maximum percentage response for somatic embryos induction (64.56%) from callus clumps was obtained on MS medium fortified with BAP (0.5 mg/1) and NAA (1.0 mg/1) by Ranjan and Gaurav [27]. For proliferation, embryogenic callus was transferred to MS liquid medium containing the same hormone; 2,4-D with a small amount of NAA and subcultured at 2 weeks interval. Induction of somatic embryos different stages (globular, heart and torpedo) were observed after 2 weeks of culture. Somatic embryos were developed in MS suspension medium containing 1.0 to 2.0 mg/l 2,4-D with 0.1 or 1.0 mg/l NAA and the globular embryos were further differentiated into the cotyledonary phase embryos. The addition of 5.0 mg/l amino acids (L-glutamine or L-proline) to the culture media, in the range of the tested medium condition, yielded higher enhancement of the embryo growth and development. Transfering of individual embryos onto a fresh basal MS medium without plant growth regulators enabled the achievement of complete maturation. Relatively, only a small number of the induced embryos developed shoots and roots when they were transferred to MS medium supplemented with 2.0 mg/l BAP and 0.5 mg/l NAA in addition of 3% (w/v) sucrose and 0.8% (w/v) agar. Nearly, 11% of somatic embryos were able to be converted to fertile plants. This similar result was supported by other authors The *in vitro* regeneration of the carnation plant species was established from aseptic shoots produced from surface sterilized seeds grown in basal MS medium. High percentage of grown seeds was obtained (50-90%) after 3 weeks of culturing the seeds. All the plantlets were rooted efficiently on the multiplication MS medium without hormone supplementation. This may be due to the presence of endogenous auxins which causes rooting as it was stated by Mosleh et al. [56]. After 4 weeks, full grown plantlets were transferred to sterile soil at ratio of 1:1:1 for garden soil, sand and loam for acclimatization process. They were maintained in the culture room at 25±1°C with 16 hours light and 8 hours dark planted in pots and covered with polystyrene plastic bags. The growth was encouraging and after 4 weeks in the culture room, well grown and healthy plantlets were transferred to the greenhouse. Survival rate of the acclimatized plantlets was 78% and it showed that the most of the plantlets successfully survived after transferred to the greenhouse. Best response for hardening of the plant species was also obtained by Aamir et al. [57] (95%) in mixture containing sand, peat and soil (1:1:1) under natural light conditions [58].

#### *6.2.4. Saintpaulia ionantha*

The complete plant regeneration was obtained from leaf, petiole, peduncle and floral parts of the plant. For shoot regeneration, MS medium supplemented with IAA (1.0 mg/l) and Zeatin (2.0 mg/l) induced the highest number of shoots (15.0) in 8 weeks. While shoots regeneration and multiplication obtained from the same plant by Azura et al. [59] with maximum induction rate on MS medium supplemented with 3.0 mg/l BAP and 1.0 mg/l of NAA. It was reported by Hasbullah et al. [10] that a good combination of cytokinins and auxin in the plant culture medium enhanced good shoot formation and plantlet regeneration. Most of the developed plantlets were hardened (84%) and acclimatized in the greenhouse by transferring the plantlets into planting pots containing mixed soil, which is a mixture of compost, sand and black soil with the ratio of 1:1:2. Meanwhile, Khan et al. found that among the different potting mixes used for the acclimatization of rooted plantlets, 100% sand was found to be the best [59].

However, the obtained new plants failed to flower even after twelve months from planting. The morphological characters of these plantlets were compared to those of the mother plants. They were found to be different in some of their morphological characters such as plant height, leaf size and leaf texture and showed similarity in leaf arrangement and leaf margin and they showed unstable morphological characters. But variations in flowering period, number of flowers per plant and flower morphology were observed by Jain [17] in the plants directly regenerated from leaf disk explants. So, he concluded that the cytokinins, benzylaminopurine and zeatin tested in the culture medium did not affect the basic plant characteristics including flower colour which remained stable in both species. An attempt to induce *in vitro* flowering from african violet was also reported by Daud and Taha [9]. They found that the floral buds were formed *in vitro* with sepal (calyx) and petal (corolla) but did not show any formation of reproductive organs (stamens or pistils).

## **6.3. Acclimatization**

Acclimatization process were carried out while the plants still under *in vitro* condition. A few days before the process was to be carried out, the cover of test tube was removed. With the relative humidity at 50-70% in the culture room, this will increase the epicuticular wax development on the upper leaf surfaces of the plantlets and their survival rate rose from 70 to 90% [60]. The plantlets were exposed to the normal environment in stages as they will wilt due to rapid changes of relative humidity and light intensity. *In vitro* plantlets that reached 3-5 cm height were taken out from culture tubes and the excess media were rinsed to avoid contamination. They were then put into plastic pots and planted out in soil at a ratio of 1:1:1 for garden soil, sand and loam. There were 3 types of treatments which were carried out:

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 267

**Inhibition Zone of Tested Bactria (mm) E.c. P.a. S.a. B.c.** 

Meuller Hinton agar, Meuller Hinton Broth, Sabouraud Dextrose Agar (Difco) and Sabouraud Dextrose Broth (Difco) culture media were used. Each medium was prepared to manufacturers` specification and adjusted to the appropriate pH before sterilized by autoclaving at 121 C for 15 minutes. About 20 ml sterile agar media were poured into petri dishes and let to solidify at a slanted position in Universal bottles. Broth or liquid media were distributed into final containers before autoclaving. Antioxidant and antibacterial activities of ethanolic extracts of *Asparagus officinalis* cv. Mary Washington grown *in vivo* and *in vitro* were compared in our laboratory [61]. Although no antibacterial activity was detected from both *in vivo* and *in vitro* grown plant extracts in the disc diffusion antimicrobial assay, ethanolic extract of *A. officinalis* offered antibacterial activity against

*Zizyphus jujube* - 0.67±0.29 0.5±0.0 - *Thymus vulgaris* L. 0.93±0.12 0.5±0.0 2.33±0.58 1.17±0.29 *Carum carvi* L. - 0.5±0.0 0.83±0.29 - *Teucrium polium* L. - 0.5±0.0 1.0±0.0 - *Althaea officinalis* L. - 0.5±0.0 1.5±0.5 - *Borage officinalis* L - - - - Tetracycline (30μg) 13±0.1 2.97±0.06 12.97±0.06 8.07±0.12 **Table 1.** Inhibition effect of 100 mg/ml of ethanolic extracts of some ornamental plants against the

**Figure 1.** Comparison of antibacterial activity of *in vitro, in vivo* and callus extracts of *Asparagus officinalis*

against *Bacillus cerus* and *Pseudomonas aeruginosa*, using agar diffusion method

*6.4.1. Media for microbial cultivation and maintenance* 

*Bacillus cereus* (Table 2; Figure 1).

growth of four pathogenic bacteria.

B.c: Bacillus cereus; P.a: Pseudomonas aeruginosa

**Plant name** 


For acclimatization purpose of (*Saintpaulia ionantha*), various substrates were used, such as autoclaved mixed soil (compost, sand, and black soil in the ratio 1:1:2) and non-autoclaved mixed soil. The regenerated plants must reach 4-5 cm before transferring them into pots of mixed soil. After transplanting, the plantlets were watered regularly to prevent from drying. For the first 3 weeks the regenerated plants were maintained in the culture room at 25±2 °C. Gamma irradiations of 10-60 gray were also tested on the regenerated plants to induce flowering and also to observe the effect of radiation on the plantlets. Successful micropropagation of plants which can survive under the natural environmental conditions depends on acclimatization process. Most species grown *in vitro* required an acclimatization process in order to ensure that sufficient number of plants can survive and grow vigorously after being transferred to ex *vivo* soil. The excess media was cleaned from the roots and the plants were transplanted in an adequate substrate such as peat or soil. Plantlets were maintained in a confined environment temporarily before they can be adapted progressively in typical environment within drier air, high light intensity and temperature variations.

#### **6.4. Antimicrobial studies**

To determine the antimicrobial activity, *Pereskia grandifolia* fresh leaves (300 g) were dried in oven (30-35 °C) for about 5-7 days. Dried leaves were crushed and ground using mortar and pestle in the laboratory. The final weights of the dried powdered materials were 35 g. The leaf powder was extracted with methanol as a solvent. Extracts were filtered and concentrated to dryness using a rotary evaporator. Extract was then ready for the antimicrobials tests. The same procedure was done with aseptic callus to obtain the extract of callus. The antimicrobial screening battery consisted of: gram positive bacterium, *Bacillus subtilis*, *Staphylococcus aureus*; gram negative bacteria, *Escherichia coli*, *Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae* and *Pseudomonas aeruginosa;* and Fungi, *Candida albicans, Microsporium cants*, *Trichophyton rubrum, Trichophyton mentagrophyte* and *Aspergillus niger.* The antimicrobials tests were done using standard microbial test culture.

#### *6.4.1. Media for microbial cultivation and maintenance*

266 Plant Science

carried out:

**6.4. Antimicrobial studies** 

**6.3. Acclimatization** 

Acclimatization process were carried out while the plants still under *in vitro* condition. A few days before the process was to be carried out, the cover of test tube was removed. With the relative humidity at 50-70% in the culture room, this will increase the epicuticular wax development on the upper leaf surfaces of the plantlets and their survival rate rose from 70 to 90% [60]. The plantlets were exposed to the normal environment in stages as they will wilt due to rapid changes of relative humidity and light intensity. *In vitro* plantlets that reached 3-5 cm height were taken out from culture tubes and the excess media were rinsed to avoid contamination. They were then put into plastic pots and planted out in soil at a ratio of 1:1:1 for garden soil, sand and loam. There were 3 types of treatments which were

For acclimatization purpose of (*Saintpaulia ionantha*), various substrates were used, such as autoclaved mixed soil (compost, sand, and black soil in the ratio 1:1:2) and non-autoclaved mixed soil. The regenerated plants must reach 4-5 cm before transferring them into pots of mixed soil. After transplanting, the plantlets were watered regularly to prevent from drying. For the first 3 weeks the regenerated plants were maintained in the culture room at 25±2 °C. Gamma irradiations of 10-60 gray were also tested on the regenerated plants to induce flowering and also to observe the effect of radiation on the plantlets. Successful micropropagation of plants which can survive under the natural environmental conditions depends on acclimatization process. Most species grown *in vitro* required an acclimatization process in order to ensure that sufficient number of plants can survive and grow vigorously after being transferred to ex *vivo* soil. The excess media was cleaned from the roots and the plants were transplanted in an adequate substrate such as peat or soil. Plantlets were maintained in a confined environment temporarily before they can be adapted progressively in typical environment within drier air, high light intensity and temperature variations.

To determine the antimicrobial activity, *Pereskia grandifolia* fresh leaves (300 g) were dried in oven (30-35 °C) for about 5-7 days. Dried leaves were crushed and ground using mortar and pestle in the laboratory. The final weights of the dried powdered materials were 35 g. The leaf powder was extracted with methanol as a solvent. Extracts were filtered and concentrated to dryness using a rotary evaporator. Extract was then ready for the antimicrobials tests. The same procedure was done with aseptic callus to obtain the extract of callus. The antimicrobial screening battery consisted of: gram positive bacterium, *Bacillus subtilis*, *Staphylococcus aureus*; gram negative bacteria, *Escherichia coli*, *Proteus mirabilis, Proteus vulgaris, Klebsiella pneumoniae* and *Pseudomonas aeruginosa;* and Fungi, *Candida albicans, Microsporium cants*, *Trichophyton rubrum, Trichophyton mentagrophyte* and *Aspergillus* 

*niger.* The antimicrobials tests were done using standard microbial test culture.

1. Plantlets were planted in a pot and placed a beaker to cover it up.

3. Plantlets were planted in a pot and covered with transparent plastic lid.

2. Plantlets were planted in a pot and put in Mistifier device.

Meuller Hinton agar, Meuller Hinton Broth, Sabouraud Dextrose Agar (Difco) and Sabouraud Dextrose Broth (Difco) culture media were used. Each medium was prepared to manufacturers` specification and adjusted to the appropriate pH before sterilized by autoclaving at 121 C for 15 minutes. About 20 ml sterile agar media were poured into petri dishes and let to solidify at a slanted position in Universal bottles. Broth or liquid media were distributed into final containers before autoclaving. Antioxidant and antibacterial activities of ethanolic extracts of *Asparagus officinalis* cv. Mary Washington grown *in vivo* and *in vitro* were compared in our laboratory [61]. Although no antibacterial activity was detected from both *in vivo* and *in vitro* grown plant extracts in the disc diffusion antimicrobial assay, ethanolic extract of *A. officinalis* offered antibacterial activity against *Bacillus cereus* (Table 2; Figure 1).


**Table 1.** Inhibition effect of 100 mg/ml of ethanolic extracts of some ornamental plants against the growth of four pathogenic bacteria.

B.c: Bacillus cereus; P.a: Pseudomonas aeruginosa

**Figure 1.** Comparison of antibacterial activity of *in vitro, in vivo* and callus extracts of *Asparagus officinalis* against *Bacillus cerus* and *Pseudomonas aeruginosa*, using agar diffusion method


*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 269

methanol (MeOH) as blank disks were used as negative controls. Incubation of bacteria and fungi were done at 37°C for 24 hrs. Each extract and control was employed in triplicate for each organism. Diameters of clear zones produced around the disks (if present) were measured after the incubation time. This study showed that the antioxidant and antimicrobial activities showed that these bioactivities differ between *in vitro* and *in vivo* grown plants. Total antioxidant capacity of *in vivo* grown plant was higher than *in vitro* grown plant, while the only antimicrobial activity was obtained from *in vitro* callus tissue against ornamental plants. Some phytochemical studies are required to investigate the production of antioxidant and antimicrobial compounds in differentiated and

Arnone et al. and Drouhet et al. reported the conidial suspensions of fungi test were diluted 10 times with molten Sabouraud Dextrose Agar at 40 °C and 20 ml was poured into each petri dish [63, 64]. Filter paper disks (Whatman No. 1, 6 mm in diameter) were impregnated with the test extract solutions in methanol (MeOH) at the concentration of 100, 200, 400 and 800 mg/ml. The disks were transferred into the surface of solidified agar after evaporation of solvent. 5-Fluorocytosine at the concentration of 1.0 mg/ml, saturated filter paper disks of methanol as blank disks were applied as controls. Three series of determination were run for each extract and species of fungi. Zones of growth inhibition were measured after 3 days incubation at 37°C for *Aspergillus niger,* whereas a week at room temperature (27 °C) for *Trichophyton mentagraphyte* and *Trichophyton rubrum.* The results of well diffusion antimicrobial activity carried out and showed that the ethanolic extract of *A. graveolens* callus inhibited the growth of *Bacillus subtitles* more than ethanolic extract of the plant roots. Antimicrobial activity of the callus extract might be either related to the production of a compound in only undifferentiated callus cells or may be produced in higher amounts in these cells when compared to differentiated cells. Several quantitative estimations and studies showed that the production of biocompounds can vary between differentiated and

 The development of culture methods, particularly those far highly sealing plants, is expected to have a significant environmental benefit by controlling to retention of

The ornamental and medicinal plants demand for raw materials raises questions

 Development of *in vitro* culture technology is of fundamental importance if ornamental biotechnology is to play a central role in the growth of global ornamental plant

 Ornamental plant tissue culture techniques are expected to be developed enough in the near future when combined with molecular genetics. This may give support to be same

surrounding the sustainability of the new industry.

undifferentiated callus cells of ornamental plants.

*6.4.4. Agar diffusion assay* 

undifferentiated plant cells [50,65].

production industry in future.

**6.5. Future research** 

biodiversity.


**Table 2.** Inhibition effect of 100 mg/ml of *Asparagus officinalis* ethanolic extracts (*in vivo* plant, *in vitro*  plant and callus) against the growth of four pathogenic bacteria

#### *6.4.2. Inoculums for antimicrobial testing*

Cultures of fungi and bacteria grown in Mueller Hinton Broth and Sabouraud Dextrose Broth for 18 hours, respectively, were standardized to an optical density of 1 at 600 nm (OD600 = 1) using NOVASPEC II Visible Spectrophotometer. The density was adjusted by adding sterile broth to the cultures. The concentrations of the resultant suspensions of fungi, bacteria and yeasts were approximately 108 cells/ml and 107 cells/ml, respectively [62]. The fungi and bacteria suspensions were prepared immediately before carrying out the antimicrobial assay. Conidial suspensions of the test fungi were prepared by pouring 20 ml broth containing 1 drop Tween 80 (Sigma P-8074), into 3-days-old cultures of *Aspergillus* species or 2-week-old culture of *Trichophyton mentagraphytes.* The culture of *Aspergillus* species and *Trichophyton mentagraphytes* were grown on Sabouraud Dextrose Agar slants at 37 C and 27 C, respectively. After homogenizing with glass beads, the concentrations of the resultant conidial suspensions of *Aspergillus* species and *Trichophyton mentagraphytes* were adjusted to 106 conidia/ml and 108 conidia/ml, respectively by using haemocytometer.

#### *6.4.3. Semi-quantitative antimicrobial activity test*

According to paper-disk Diffusion assay method, the suspension culture of fungi and bacteria were diluted to the final concentrations of approximately 106 cells/ml and 105 cells/ml, respectively. The bacterial suspensions were evenly spread on the surface of 4 mm thickness of Meuller Hinton Agar (MHA) plate and the fungi suspensions on Dextrose Sabouraud Agar (DSA) plates. Sterile cotton swabs were used to produce uniform growth of organism. Methanol (MeOH) extracts of the leaves (*in vivo*) and callus (*in vitro*) of *Pereskia grandifolia* were used as test extracts. These extracts were dissolved in methanol and applied to filter disks (Whatman No 1, 6 mm in diameter), at the concentrations of 100, 200, 400 and 800 mg/ml for fungi, yeasts and antibacterial screening. After evaporation of the solvent, the disks were placed in a good contact on the seeded agar plates. Chloramphenicol and 5 fluorocytosine (SIGMA F-7129) at the concentrations of the 1.0 mg/ml were used as positive controls for antifungal and antibacterial, respectively. Saturated filter paper disk of methanol (MeOH) as blank disks were used as negative controls. Incubation of bacteria and fungi were done at 37°C for 24 hrs. Each extract and control was employed in triplicate for each organism. Diameters of clear zones produced around the disks (if present) were measured after the incubation time. This study showed that the antioxidant and antimicrobial activities showed that these bioactivities differ between *in vitro* and *in vivo* grown plants. Total antioxidant capacity of *in vivo* grown plant was higher than *in vitro* grown plant, while the only antimicrobial activity was obtained from *in vitro* callus tissue against ornamental plants. Some phytochemical studies are required to investigate the production of antioxidant and antimicrobial compounds in differentiated and undifferentiated callus cells of ornamental plants.

#### *6.4.4. Agar diffusion assay*

268 Plant Science

**Bacteria** 

significantly different in each row (p < 0.05).

*6.4.2. Inoculums for antimicrobial testing* 

plant and callus) against the growth of four pathogenic bacteria

*6.4.3. Semi-quantitative antimicrobial activity test* 

**Inhibition Zone (mm)**

**Callus Tetracycline** 

**(30 µg)** 

*In Vitro* **Plant**

*Escherichia coli* -b -b -b 42 ± 3.00a *Staphylococcus aureus* -b -b -b 20 ± 2.64a *Pseudomonas aeruginosa* -b -b -b 11 ± 2.00a *Bacillus cereus* -c -c 12 ± 1.00b 40 ± 3.60a -: No inhibition; The data were analyzed by one-way ANOVA and the inhibition means of samples were compared using Duncan's Multiple Comparison Test (DMCT). Mean of different samples labeled with different letters are

**Table 2.** Inhibition effect of 100 mg/ml of *Asparagus officinalis* ethanolic extracts (*in vivo* plant, *in vitro* 

Cultures of fungi and bacteria grown in Mueller Hinton Broth and Sabouraud Dextrose Broth for 18 hours, respectively, were standardized to an optical density of 1 at 600 nm (OD600 = 1) using NOVASPEC II Visible Spectrophotometer. The density was adjusted by adding sterile broth to the cultures. The concentrations of the resultant suspensions of fungi, bacteria and yeasts were approximately 108 cells/ml and 107 cells/ml, respectively [62]. The fungi and bacteria suspensions were prepared immediately before carrying out the antimicrobial assay. Conidial suspensions of the test fungi were prepared by pouring 20 ml broth containing 1 drop Tween 80 (Sigma P-8074), into 3-days-old cultures of *Aspergillus* species or 2-week-old culture of *Trichophyton mentagraphytes.* The culture of *Aspergillus* species and *Trichophyton mentagraphytes* were grown on Sabouraud Dextrose Agar slants at 37 C and 27 C, respectively. After homogenizing with glass beads, the concentrations of the resultant conidial suspensions of *Aspergillus* species and *Trichophyton mentagraphytes* were adjusted to 106 conidia/ml and 108 conidia/ml, respectively by using haemocytometer.

According to paper-disk Diffusion assay method, the suspension culture of fungi and bacteria were diluted to the final concentrations of approximately 106 cells/ml and 105 cells/ml, respectively. The bacterial suspensions were evenly spread on the surface of 4 mm thickness of Meuller Hinton Agar (MHA) plate and the fungi suspensions on Dextrose Sabouraud Agar (DSA) plates. Sterile cotton swabs were used to produce uniform growth of organism. Methanol (MeOH) extracts of the leaves (*in vivo*) and callus (*in vitro*) of *Pereskia grandifolia* were used as test extracts. These extracts were dissolved in methanol and applied to filter disks (Whatman No 1, 6 mm in diameter), at the concentrations of 100, 200, 400 and 800 mg/ml for fungi, yeasts and antibacterial screening. After evaporation of the solvent, the disks were placed in a good contact on the seeded agar plates. Chloramphenicol and 5 fluorocytosine (SIGMA F-7129) at the concentrations of the 1.0 mg/ml were used as positive controls for antifungal and antibacterial, respectively. Saturated filter paper disk of

*In Vivo* **Plant**

> Arnone et al. and Drouhet et al. reported the conidial suspensions of fungi test were diluted 10 times with molten Sabouraud Dextrose Agar at 40 °C and 20 ml was poured into each petri dish [63, 64]. Filter paper disks (Whatman No. 1, 6 mm in diameter) were impregnated with the test extract solutions in methanol (MeOH) at the concentration of 100, 200, 400 and 800 mg/ml. The disks were transferred into the surface of solidified agar after evaporation of solvent. 5-Fluorocytosine at the concentration of 1.0 mg/ml, saturated filter paper disks of methanol as blank disks were applied as controls. Three series of determination were run for each extract and species of fungi. Zones of growth inhibition were measured after 3 days incubation at 37°C for *Aspergillus niger,* whereas a week at room temperature (27 °C) for *Trichophyton mentagraphyte* and *Trichophyton rubrum.* The results of well diffusion antimicrobial activity carried out and showed that the ethanolic extract of *A. graveolens* callus inhibited the growth of *Bacillus subtitles* more than ethanolic extract of the plant roots. Antimicrobial activity of the callus extract might be either related to the production of a compound in only undifferentiated callus cells or may be produced in higher amounts in these cells when compared to differentiated cells. Several quantitative estimations and studies showed that the production of biocompounds can vary between differentiated and undifferentiated plant cells [50,65].

#### **6.5. Future research**


biotechnological applications as in ornamental plant in genomic age, a field in which ornamental plant is also far behind other higher plants.

*In vitro* Regeneration, Acclimatization and Antimicrobial Studies of Selected Ornamental Plants 271

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## **7. Conclusion**

Successful *in vitro* propagation, organogenesis and somatic embryogenesis with acclimatization of ornamental plants is now being used for commercialization. Many commercial laboratories and national research institutes worldwide use *in vitro* culture system for rapid plant multiplication, germplasm conservation, elimination of pathogens, genetic manipulations and secondary metabolite production. This somatic embryos protocol could be useful for conservation and agronomy and in the improvement of ornamental plants using gene transfer biotechnologies. The results of present investigation clearly indicate that antimicrobial activity vary with the plant species of the ornamental plants and plant material used. Thus, the study ascertains the value of ornamental plants used in Ayurveda, which could be of considerable interest to the development of new drugs.

### **Author details**

A. Bakrudeen Ali Ahmed, S. Mohajer, E.M. Elnaiem and R.M. Taha *Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia* 

### **Acknowledgement**

The authors thank University of Malaya, Malaysia for the financial support and facilities provided.

#### **8. References**


[5] Castillo B, Smith M.A.L. Direct somatic embryogenesis from *Begonia gracilis* explants. Plant Cell report 1997*,* 16, 385-388.

270 Plant Science

biotechnologies.

**7. Conclusion** 

**Author details** 

**Acknowledgement** 

provided.

**8. References** 

biotechnological applications as in ornamental plant in genomic age, a field in which

 Thus intensive work on new strain selection and improvement of an efficient mass culture system is clearly needed. For the exploitation of ornamental plants are developed at the cellular level, plant tissue culture constituents a basic powerful tool. This efficient somatic embryogenesis protocols could be useful for conservation and agronomy and in the improvement of ornamental plants using gene transfer

Successful *in vitro* propagation, organogenesis and somatic embryogenesis with acclimatization of ornamental plants is now being used for commercialization. Many commercial laboratories and national research institutes worldwide use *in vitro* culture system for rapid plant multiplication, germplasm conservation, elimination of pathogens, genetic manipulations and secondary metabolite production. This somatic embryos protocol could be useful for conservation and agronomy and in the improvement of ornamental plants using gene transfer biotechnologies. The results of present investigation clearly indicate that antimicrobial activity vary with the plant species of the ornamental plants and plant material used. Thus, the study ascertains the value of ornamental plants used in

Ayurveda, which could be of considerable interest to the development of new drugs.

*Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia* 

The authors thank University of Malaya, Malaysia for the financial support and facilities

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[52] Taha R.M, Daud N, Hasbullah N.A. Establishment of efficient Regeneration System, Acclimatization and Somaclonal variation in *Saintpaulia ionantha H. Wendl.* Acta

[53] Dodeman V.L, Ducreux G, Kreis M. Zygotic embryogenesis versus somatic

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[55] Ahmed A.B.A, Rao A.S, Rao M.V. Somatic embryogenesis and plant regeneration from cell suspension culture of *Gymnema sylvestre* (Retz). R.Br. Ex. Roemer & Schultes.

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[22] Ziv M. *In vitro* hardening and acclimatization of tissue culture plants. In: Withers, L.A. and Alderson, P.G. (eds.), Plant Tissue Culture and its Agricultural Applications 1986.

[23] Peak D.E, Cumming B.G. *In vitro* propagation of *Begonia×tuberhybrida* from leaf sections.

[24] Eide A.K, Munster C, Heyerdahl P.H, Lyngved R, Olsen O.A.S. Liquid culture systems

[25] Wated A.A, Raghothama K.G, Kochba M, Nissim A, Gaba V. Micropropagation of *Spathiphyllum* and *Syngonium* is facilitated by use of Interfacial membrane rafts.

[26] Joseph D, Martin K.P, Madassery J, Philip V.J. *In vitro* propagation of three commercial cut flower cultivars of *Anthurium andraeanum*. Horticulture Indian Journal Experiment

[27] Ranjan S, Gaurav S. Somatic embryogenesis in Gerbera (*Gerbera jamesonii Bolus ex Hooker f.*) as influenced by explants. Journal of Ornamental Horticulture 2005*,* 8, 128-130. [28] Ahmed A.B.A, Rao A.S, Rao M.V, Taha R.M. Production of gymnemic acid depends on medium, explants, PGRs, color lights, temperature, photoperiod and sucroses in batch

[30] Dijkshoorn-Dekker M.W.C. The influence of light and temperature on the dynamic

[31] Deshpande S.R, Josekutty P.C, Prathapasenan G. Plant regeneration from axillary buds

[32] Demiralay A, Yalcin-Mendi Y, Aka-kacar Y, Cetiner S. *In vitro* propagation of *Ficus carica* L. var. *Bursa siyahi* through meristem culture. Acta Horticulturae 1998*,* 480, 165-

[33] Kumar V, Radha A, Kumar C.S. *In vitro* plant regeneration of fig (*Ficus carica* L. cv. Gular) using apical buds from mature trees. Plant Cell Report 1998, 17, 717-720. [34] Nagaraju S, Reddy S.K, Farook S.A. Propagation of *Ficus reliosa* L. from maxillary buds

[35] Murch S.J, Victor J.M.R, Saxena P.K. Auxin, calcium and sodium in somatic embryogenesis of african violet (*Saintpaulia ionantha* Wendl.) cv. Benjamin. Acta

[36] Winkelmann T, Hohe A, Schwenkel H.G. Establishing embryogenic suspension cultures in *cyclamen persicum* "Purple Flamed". Advance Plant Science 1998*,* 12, 25-30. [37] Hohe A, Winkelmann T, Schwenkel H.G. Development of somatic embryos of *Cyclamen* 

[38] Kumari M, Varghese T.M, Mehta P.K. Micropropagation of *Chrysanthemum* through shoot apex culture in two named varieties viz. Miss Universe and Snow Ball. Annals of

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[56] Mosleh M.S, Duhoky M, Salman A, Media E.M.A. *Micropropagation of carnation* (*Dianthus caryophyllus L*.). Journal of Duhok University 2009, 12, Pp. 61-66.

**Chapter 12** 

© 2012 Mostafa, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Mostafa, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

There is a current worldwide interest in finding new and safe antioxidants from natural sources such as plant material to prevent oxidative deterioration of food and to minimize oxidative damage to living cells [1]. Microalgae are photosynthetic microorganisms that are able to rapidly generate biomass from solar energy, CO2 and nutrients in bodies of water. This biomass consists of important primary metabolites such as sugars, oils and lipids, for which process path-ways exist for the production of high-value products including human and animal feed supplements, transport fuels, industrial chemicals and pharmaceuticals. Algal biomass and algae-derived compounds have a very wide range of potential applications, from animal feed and aquaculture to human nutrition and health products. Some algae are considered as rich sources of natural antioxidants. Although macroalgae have received much attention as potential natural antioxidants [2]. Furthermore, the qualities of the microalgal cells can be controlled, so that they contain no herbicides and pesticides, or any other toxic substances, by using clean nutrient media for growing the microalgae. The value of microalgae as a source of natural antioxidants is further enhanced by the relative ease of purification of target compounds. Reports on the antioxidant activity of microalgae are limited. Because cyanobacteria are largely unexplored, they represent a rich opportunity for discovery; the expected rate of rediscovery is far lower than for other better- studied groups of organisms Li et al. 2007 [3]. In this chapter, we focus on many desirable chemicals are the products of secondary metabolism triggered under conditions not conducive to fast growth. For those chemicals to be produced by microalgae, one needs to develop new strains (faster growth, higher substrate tolerance, etc.) by classical selection or genetic manipulation so microalgal biomass can be produced consistently. Highlight the role of dietary antioxidants and their potential benefits in health and disease directly or indirectly by the plant nutrition and animal feed to produce healthy organic food. Investigate the different biological activities of algae and the relations with its biochemical

**Microalgal Biotechnology:** 

**Prospects and Applications** 

Additional information is available at the end of the chapter

Soha S.M. Mostafa

http://dx.doi.org/10.5772/53649

**1. Introduction** 


**Chapter 12** 

## **Microalgal Biotechnology: Prospects and Applications**

Soha S.M. Mostafa

274 Plant Science

2008, 40, 111-121.

2007, 39, 1263-1268.

(49), 8460-8466.

1991, 2, 199-203.

Acta Horticulturae 2010, 865, 397-400.

[56] Mosleh M.S, Duhoky M, Salman A, Media E.M.A. *Micropropagation of carnation*

[57] Aamir A, Humera A, Shhagufta N, Mamoona R, Javed I. An efficient protocol for *in vitro* propogation of carnation (*Dianthus caryophyllus*). Pakestanian Journal of Botany

[58] Azura H.B.A, Taha R.M, Hasbullah N.A. Acclimatization of *Dianthus caryophyllus Linn*.

[59] Khan S, Naseeb S, Ali K. Callus induction, plant regeneration and acclimatization of African violet (*Saintpaulia ionantha*) using leaves as explants. Pakistan Journal of Botany

[60] Ziv M. Bioreactor technology for plant micropropagation. In: Janick. J. editor. Horticulture reviews, Vol. 24. New York: John Wiley & Sons Inc. 2000. Pp. 1–30. [61] Khorasani A, Sani W, Philip K, Taha R.M, Rafat A. Antioxidant and antibacterial activities of ethanolic extracts of *Asparagus officinalis* cv. Mary Washington: Comparison of *in vivo* and *in vitro* grown plant bioactivities. African Journal of Biotechnology 2010, 9

[62] Rahalison L, Hamburger M, Hostettman K. A bioautographic agar overlay method for the detection of antifungal compounds from higher plants. Phytochemical Analysis

[63] Arnone A, Assante G, Montorsi M, Nisim G. Asteromine - A bioactive secondary metabolite from a strain of *Mycospharella asteroma*. Phytochemistry 1994, 38, 595-597. [64] Drouhet E, Dupont B, Improvisi L, Viviani M.A, Tortorano A.M. Disk agar diffusion and microplate automatized technics for *in vitro* evaluation of antifungal agents on yeast and sporulated pathogenic fungi. In: Iwata, K. and Vanden Bossche, H. (eds.), proceeding of the international symposium on *in vitro* and *in vivo* evaluation of

[65] Ahmed A.B.A, Rao A.S, Rao M.V. *In vitro* production of gymnemic acid from *Gymnema sylvestre* (Retz) R.Br. Roemer and Schultes through callus culture under stress

antifungal agents held in Tokyo (Japan), 1985. Pp.19-22.

conditions. Methods Molecular Biology 2009a. 547, 93-105.

(*Dianthus caryophyllus L*.). Journal of Duhok University 2009, 12, Pp. 61-66.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53649

## **1. Introduction**

There is a current worldwide interest in finding new and safe antioxidants from natural sources such as plant material to prevent oxidative deterioration of food and to minimize oxidative damage to living cells [1]. Microalgae are photosynthetic microorganisms that are able to rapidly generate biomass from solar energy, CO2 and nutrients in bodies of water. This biomass consists of important primary metabolites such as sugars, oils and lipids, for which process path-ways exist for the production of high-value products including human and animal feed supplements, transport fuels, industrial chemicals and pharmaceuticals. Algal biomass and algae-derived compounds have a very wide range of potential applications, from animal feed and aquaculture to human nutrition and health products. Some algae are considered as rich sources of natural antioxidants. Although macroalgae have received much attention as potential natural antioxidants [2]. Furthermore, the qualities of the microalgal cells can be controlled, so that they contain no herbicides and pesticides, or any other toxic substances, by using clean nutrient media for growing the microalgae. The value of microalgae as a source of natural antioxidants is further enhanced by the relative ease of purification of target compounds. Reports on the antioxidant activity of microalgae are limited. Because cyanobacteria are largely unexplored, they represent a rich opportunity for discovery; the expected rate of rediscovery is far lower than for other better- studied groups of organisms Li et al. 2007 [3]. In this chapter, we focus on many desirable chemicals are the products of secondary metabolism triggered under conditions not conducive to fast growth. For those chemicals to be produced by microalgae, one needs to develop new strains (faster growth, higher substrate tolerance, etc.) by classical selection or genetic manipulation so microalgal biomass can be produced consistently. Highlight the role of dietary antioxidants and their potential benefits in health and disease directly or indirectly by the plant nutrition and animal feed to produce healthy organic food. Investigate the different biological activities of algae and the relations with its biochemical

© 2012 Mostafa, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Mostafa, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

composition, pigments and different constituents which may vary with salt stressed culture conditions and describe the antioxidant characteristics of algae.

Microalgal Biotechnology: Prospects and Applications 277

**Figure 1.** Main pathways of some secondary and primary metabolites biosynthesis modified from Burja

there are several classes including alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids Phytochemicals are naturally occurring, nonnutritive chemicals. They appear to work alone and in combination, and

Microalgae are significant resource for bioactive metabolites, particularly cytotoxic agents with applications in cancer chemotherapy. From the marine microalgae such as from the blooms of *Phaeocystis* sp., antibiotic substances were listed. *Phaeocystis pouchetii* is reported to produce chemicals such as acrylic acid, which constitutes about 7.0% of the dry weight. The antibiotic substances thus produced are transferred throughout the food chain and found in the digestive tract of *Antartic penguins*. Production of ß carotene and vitamins by the halotolerant alga *Dunaliella* sp. is documented. These compounds have much importance for the Mariculture activities [9]. Cyanobacteria have been identified as one of the most promising group of organisms from which novel and biochemically active natural products are isolated. Cyanobacteria such as *Spirulina*, *Anabaena*, *Nostoc* and *Oscillatoria* produce a great variety of secondary metabolites. Cyanobacteria produce a wide variety of bioactive compounds, which include 40% lipopeptides, 5.6% amino acids, 4.2% fatty acids, 4.2% macrolides and 9% amides. Cyanobacterial lipopeptides include different compounds like cytotoxic (41%), antitumor (13%), antiviral (4%), antibiotics (12%) and the remaining 18% activities include antimalarial, antimycotics, multi-drug resistance reversers, antifeedant, herbicides and immunosuppressive agents [7]; besides the immune effect, blue green algae improves metabolism. Cyanobacteria are also known to produce antitumor, antiviral,

et al. [7]

perhaps in conjunction, with vitamins [8].

**5. Microalgal bioactive compounds** 

## **2. What are microalgae?**

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that produce carbohydrates, proteins and lipids as a result of photosynthesis. They can grow rapidly and live in harsh conditions due to their unicellular or simple multicellular structure. Examples of prokaryotic microorganisms are Cyanobacteria (Cyanophyceae) and eukaryotic microalgae are for example green algae (Chlorophyta) and diatoms (Bacillariophyta). Microalgae are present in all existing earth ecosystems, not just aquatic but also terrestrial, representing a big variety of species living in a wide range of environmental conditions. It is estimated that more than 50,000 species exist, but only a limited number, of around 30,000, have been studied and analyzed [4]. Sunlight, water, nutrients and arable land are the major requirements for growing algae. Micro algae have the ability to fix CO2 using solar energy with efficiency 10 times greater than that of the terrestrial plants with numerous additional technological advantages. Algae are more efficient at utilizing sunlight than terrestrial plants, consume harmful pollutants, have minimal resource requirements and do not compete with food or agriculture for precious resources [5].

## **3. Algal metabolites**

Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes, but usually has important ecological function. The induction of secondary metabolism is linked to particular environmental conditions or developmental stages. Secondary metabolites are those chemical compounds in organisms that are not directly involved in the normal growth, development or reproduction of organisms.The exploration of these organisms for pharmaceutical purposes has revealed important chemical prototypes for the discovery of new agents, stimulating the use of sophisticated physical techniques and new syntheses of compounds with biomedical application. In this regard, both secondary and primary metabolisms have been studied as a prelude to future rational economic exploitation (Figure 1). The secondary metabolism is of restricted distribution, while the primary metabolism furnishes intermediates for the synthesis of essential macromolecules [6].

## **4. What are phytochemicals?**

"Phyto" is the Greek word for plant. The term "phytochemicals" refers to a wide variety of compounds produced by plants. Phytochemicals are chemical compounds formed during the plants normal metabolic processes. There are many "families" of phytochemicals and they help the human body in a variety of ways. Phytochemicals may protect human from a host of diseases. These chemicals are often referred to as "secondary metabolities" of which

**Figure 1.** Main pathways of some secondary and primary metabolites biosynthesis modified from Burja et al. [7]

there are several classes including alkaloids, flavonoids, coumarins, glycosides, gums, polysaccharides, phenols, tannins, terpenes and terpenoids Phytochemicals are naturally occurring, nonnutritive chemicals. They appear to work alone and in combination, and perhaps in conjunction, with vitamins [8].

#### **5. Microalgal bioactive compounds**

276 Plant Science

**2. What are microalgae?** 

**3. Algal metabolites** 

essential macromolecules [6].

**4. What are phytochemicals?** 

composition, pigments and different constituents which may vary with salt stressed culture

Microalgae are prokaryotic or eukaryotic photosynthetic microorganisms that produce carbohydrates, proteins and lipids as a result of photosynthesis. They can grow rapidly and live in harsh conditions due to their unicellular or simple multicellular structure. Examples of prokaryotic microorganisms are Cyanobacteria (Cyanophyceae) and eukaryotic microalgae are for example green algae (Chlorophyta) and diatoms (Bacillariophyta). Microalgae are present in all existing earth ecosystems, not just aquatic but also terrestrial, representing a big variety of species living in a wide range of environmental conditions. It is estimated that more than 50,000 species exist, but only a limited number, of around 30,000, have been studied and analyzed [4]. Sunlight, water, nutrients and arable land are the major requirements for growing algae. Micro algae have the ability to fix CO2 using solar energy with efficiency 10 times greater than that of the terrestrial plants with numerous additional technological advantages. Algae are more efficient at utilizing sunlight than terrestrial plants, consume harmful pollutants, have minimal resource requirements and do not

Metabolites are the intermediates and products of metabolism. The term metabolite is usually restricted to small molecules. A primary metabolite is directly involved in the normal growth, development, and reproduction. A secondary metabolite is not directly involved in those processes, but usually has important ecological function. The induction of secondary metabolism is linked to particular environmental conditions or developmental stages. Secondary metabolites are those chemical compounds in organisms that are not directly involved in the normal growth, development or reproduction of organisms.The exploration of these organisms for pharmaceutical purposes has revealed important chemical prototypes for the discovery of new agents, stimulating the use of sophisticated physical techniques and new syntheses of compounds with biomedical application. In this regard, both secondary and primary metabolisms have been studied as a prelude to future rational economic exploitation (Figure 1). The secondary metabolism is of restricted distribution, while the primary metabolism furnishes intermediates for the synthesis of

"Phyto" is the Greek word for plant. The term "phytochemicals" refers to a wide variety of compounds produced by plants. Phytochemicals are chemical compounds formed during the plants normal metabolic processes. There are many "families" of phytochemicals and they help the human body in a variety of ways. Phytochemicals may protect human from a host of diseases. These chemicals are often referred to as "secondary metabolities" of which

conditions and describe the antioxidant characteristics of algae.

compete with food or agriculture for precious resources [5].

Microalgae are significant resource for bioactive metabolites, particularly cytotoxic agents with applications in cancer chemotherapy. From the marine microalgae such as from the blooms of *Phaeocystis* sp., antibiotic substances were listed. *Phaeocystis pouchetii* is reported to produce chemicals such as acrylic acid, which constitutes about 7.0% of the dry weight. The antibiotic substances thus produced are transferred throughout the food chain and found in the digestive tract of *Antartic penguins*. Production of ß carotene and vitamins by the halotolerant alga *Dunaliella* sp. is documented. These compounds have much importance for the Mariculture activities [9]. Cyanobacteria have been identified as one of the most promising group of organisms from which novel and biochemically active natural products are isolated. Cyanobacteria such as *Spirulina*, *Anabaena*, *Nostoc* and *Oscillatoria* produce a great variety of secondary metabolites. Cyanobacteria produce a wide variety of bioactive compounds, which include 40% lipopeptides, 5.6% amino acids, 4.2% fatty acids, 4.2% macrolides and 9% amides. Cyanobacterial lipopeptides include different compounds like cytotoxic (41%), antitumor (13%), antiviral (4%), antibiotics (12%) and the remaining 18% activities include antimalarial, antimycotics, multi-drug resistance reversers, antifeedant, herbicides and immunosuppressive agents [7]; besides the immune effect, blue green algae improves metabolism. Cyanobacteria are also known to produce antitumor, antiviral, antifungal compounds and have a cholesterol-lowering effect in animals and humans [10]. Many of the pharmaceutically interesting compounds in cyanobacteria are peptides, including cyanobacterial toxins and important candidates for anti-cancer drugs. Peptide synthetases are common in cyanobacteria and responsible for the production of cyanobacterial hepatotoxins and other peptides. Polyketide synthetases are also involved in the biosynthesis of certain cyanobacterial bioactive compounds (e.g. microcystins). A number of extracts were found to be remarkably active in protecting human lymphoblastoid T-cells from the cytopathic effects of HIV infection. Active agents consisting of sulfolipids with different fatty acid esters were isolated from *Lyngbya lagerheimii* and *Phormidium tenue*. Cyanovirin is a protein isolated from an aqueous cellular extract of *Nostoc elipsosporum* prevents the in vitro replication and citopathicity of primate retroviruses.Cryptophycin 1, an active compound isolated from *Nostoc* strain, exerts antiproliferative and antimitotic activities by binding to the ends of the microtubules, thus blocking the cell cycle at the metaphase of mitosis. Research has been focused on its potent antitumor activity and a synthetic analogue, cryptophycin-52, is at present in Phase II clinical trials. Sulfated polysaccharide, calcium spirulan. A novel water soluble extracts of cyanobacteria have found to be an antiviral agent. This compound appears to be selectively inhibiting the penetration of enveloped viruses into host cells, thereby preventing the replication. The effect was described for many different viruses like herpex simplex, measles, and even HIV-1. Among eukaryotic microalgae, a glycoprotein prepared from *Chlorella vulgaris* culture supernatant exhibited protective activity against tumor metastasis and chemotherapyinduced immunosuppression in mice [11]. Hereafter, a brief discuss of the commercial application of the most explored compounds from algae and the biosynthetic pathways of fatty acids, steroids and carotenoids.

Microalgal Biotechnology: Prospects and Applications 279

hormone-like substances such as prostaglandins, thromboxanes and leucotrienes that are crucial in regulating developmental and regulatory physiology (Figure 2) [14]. Consumption of n-3 PUFAs from both seafood and plant sources may reduce coronary heart disease (CHD) risk as reported by Mozaffarian et al. [15] in a cohort study of 45,722 men. Thus, many health supplement stores now sell preparation of microalgae such as *Spirulina* and *Chlorella* packed in capsule or caplets, or even in food and beverages known to have therapeutic values in treating hypercholesterolemia, hyperlipidaemia and atherosclerosis [16]. The fatty acid contents of microalgae are influenced by the environmental and cultural condition selected for its growth [17]. Some of the environmental conditions include heterotrophic, photoautotrophic and nitrogen deprivation or stimulation. Although some microalgae species are cultivated as sources of these fatty acids, transgenic algae engineered to produce EPA, like transgenic oilseed crops, could provide an alternative sustainable source of oil for human consumption [18]. However, the possibility for deploying transgenic organisms nutritionally enhancedwith EPA is currently limited by continued consumer antipathy to transgenic food products. One alternative would be to use EPA from transgenic algae as a high potential food source in aquaculture. In this way, the significant health benefits of these fatty acids could be delivered into the human diet, without the requirement

**Figure 2.** A simplified biosynthesis scheme of eicosapentaenoic acid and eicosanoid (prostaglandins,

Sterols are one of the most important chemical constituents of microalgae [20]. Sterols are the main component of eukaryote organisms and different classes of organisms have divergent sterols patterns. It is because of this that sterols act as a fingerprint for organic

thromboxanes, leukotrienes) modified from Sayanova and Napier [19].

**5.2. Sterols** 

of direct ingestion of genetically modified food.

#### **5.1. Fatty Acids (FA)**

Microalgae include essential fatty acids (EFAs) such as linoleic, arachidonic, linolenic, ? linolenic acids etc. that must be in diet for healthy growth. These acids cannot be synthesized fast enough by body to meet needs [12]. Fatty acids are structural components of many lipids, and the types and amounts of fatty acids vary considerably among algae. In recent years, fatty acids compositions in large scale production of microalgae including marine algae have created considerable interest among researchers. This is mainly because of the health benefit of mono and polyunsaturated fatty acids (MUFA and PUFA) that can be found in plants including microalgae. Moreover, polyunsaturated fatty acids (PUFAs) play key roles in cellular and tissue metabolism, including the regulation of membrane fluidity, electron and oxygen transport, as well as thermal adaptation [13]. The biosynthesis of EPA occurs through a series of reactions that can be divided into two distinct steps. First is the de novo synthesis of oleic acid (18:1 ω9) from acetate, followed by conversion to linoleic acid (18:2 ω-6) and α-linolenic acid (18:3 ω-3). The subsequent stepwise desaturation and elongation steps form an ω-3 PUFA (Fig. 2). Inside the cell, EPA is normally esterified (by cyclooxygenase and lipooxygenase activities) to form complex lipid molecules and plays an important role in higher animals and humans as the precursor of a group of eicosanoids,

hormone-like substances such as prostaglandins, thromboxanes and leucotrienes that are crucial in regulating developmental and regulatory physiology (Figure 2) [14]. Consumption of n-3 PUFAs from both seafood and plant sources may reduce coronary heart disease (CHD) risk as reported by Mozaffarian et al. [15] in a cohort study of 45,722 men. Thus, many health supplement stores now sell preparation of microalgae such as *Spirulina* and *Chlorella* packed in capsule or caplets, or even in food and beverages known to have therapeutic values in treating hypercholesterolemia, hyperlipidaemia and atherosclerosis [16]. The fatty acid contents of microalgae are influenced by the environmental and cultural condition selected for its growth [17]. Some of the environmental conditions include heterotrophic, photoautotrophic and nitrogen deprivation or stimulation. Although some microalgae species are cultivated as sources of these fatty acids, transgenic algae engineered to produce EPA, like transgenic oilseed crops, could provide an alternative sustainable source of oil for human consumption [18]. However, the possibility for deploying transgenic organisms nutritionally enhancedwith EPA is currently limited by continued consumer antipathy to transgenic food products. One alternative would be to use EPA from transgenic algae as a high potential food source in aquaculture. In this way, the significant health benefits of these fatty acids could be delivered into the human diet, without the requirement of direct ingestion of genetically modified food.

**Figure 2.** A simplified biosynthesis scheme of eicosapentaenoic acid and eicosanoid (prostaglandins, thromboxanes, leukotrienes) modified from Sayanova and Napier [19].

#### **5.2. Sterols**

278 Plant Science

fatty acids, steroids and carotenoids.

**5.1. Fatty Acids (FA)** 

antifungal compounds and have a cholesterol-lowering effect in animals and humans [10]. Many of the pharmaceutically interesting compounds in cyanobacteria are peptides, including cyanobacterial toxins and important candidates for anti-cancer drugs. Peptide synthetases are common in cyanobacteria and responsible for the production of cyanobacterial hepatotoxins and other peptides. Polyketide synthetases are also involved in the biosynthesis of certain cyanobacterial bioactive compounds (e.g. microcystins). A number of extracts were found to be remarkably active in protecting human lymphoblastoid T-cells from the cytopathic effects of HIV infection. Active agents consisting of sulfolipids with different fatty acid esters were isolated from *Lyngbya lagerheimii* and *Phormidium tenue*. Cyanovirin is a protein isolated from an aqueous cellular extract of *Nostoc elipsosporum* prevents the in vitro replication and citopathicity of primate retroviruses.Cryptophycin 1, an active compound isolated from *Nostoc* strain, exerts antiproliferative and antimitotic activities by binding to the ends of the microtubules, thus blocking the cell cycle at the metaphase of mitosis. Research has been focused on its potent antitumor activity and a synthetic analogue, cryptophycin-52, is at present in Phase II clinical trials. Sulfated polysaccharide, calcium spirulan. A novel water soluble extracts of cyanobacteria have found to be an antiviral agent. This compound appears to be selectively inhibiting the penetration of enveloped viruses into host cells, thereby preventing the replication. The effect was described for many different viruses like herpex simplex, measles, and even HIV-1. Among eukaryotic microalgae, a glycoprotein prepared from *Chlorella vulgaris* culture supernatant exhibited protective activity against tumor metastasis and chemotherapyinduced immunosuppression in mice [11]. Hereafter, a brief discuss of the commercial application of the most explored compounds from algae and the biosynthetic pathways of

Microalgae include essential fatty acids (EFAs) such as linoleic, arachidonic, linolenic, ? linolenic acids etc. that must be in diet for healthy growth. These acids cannot be synthesized fast enough by body to meet needs [12]. Fatty acids are structural components of many lipids, and the types and amounts of fatty acids vary considerably among algae. In recent years, fatty acids compositions in large scale production of microalgae including marine algae have created considerable interest among researchers. This is mainly because of the health benefit of mono and polyunsaturated fatty acids (MUFA and PUFA) that can be found in plants including microalgae. Moreover, polyunsaturated fatty acids (PUFAs) play key roles in cellular and tissue metabolism, including the regulation of membrane fluidity, electron and oxygen transport, as well as thermal adaptation [13]. The biosynthesis of EPA occurs through a series of reactions that can be divided into two distinct steps. First is the de novo synthesis of oleic acid (18:1 ω9) from acetate, followed by conversion to linoleic acid (18:2 ω-6) and α-linolenic acid (18:3 ω-3). The subsequent stepwise desaturation and elongation steps form an ω-3 PUFA (Fig. 2). Inside the cell, EPA is normally esterified (by cyclooxygenase and lipooxygenase activities) to form complex lipid molecules and plays an important role in higher animals and humans as the precursor of a group of eicosanoids,

Sterols are one of the most important chemical constituents of microalgae [20]. Sterols are the main component of eukaryote organisms and different classes of organisms have divergent sterols patterns. It is because of this that sterols act as a fingerprint for organic matter input into an aquatic environment. Furthermore, sterols have a relatively high resistance to degradation when settled in anoxic sediments and persist in the environment for a longer period of time. Of all the sterol compounds, cholesterol is the most abundant and ubiquitous one in the environment, which is due to it having a variety of sources [21]. Most biologically produced sterols are planar 3-hydroxy tetracyclic structures commonly containing a methyl- or ethyl- substituted C7-C11 hydrocarbon side chain, and exhibiting a range of methyl-substitution (C4, C14) patterns on the polycyclic nucleus with varying degrees and positions of unsaturation (C5, C7, C8). The rigid structure of the sterols (Figure 3), caused by the fused ring system, provides the cell membrane integrity and stability thus, holds the membrane together. In general, there is not a specific sterol that can be uniquely linked to one algal source. Many of the sterols previously discussed are also found in other groups of algae [22].

Microalgal Biotechnology: Prospects and Applications 281

precursors) in their diet in order to acquire the adequate color for seafoodmarket acceptance [23]. Several natural sources–such as the algae *Dunaliella salina* and *Spirulina maxima*–or synthetic β-carotene, canthaxanthin and astaxanthin have been used for this purpose. Astaxanthin is, in fact, one of the most expensive components of salmon farming, accounting for about 15%of total production costs [48]. Among the several natural sources of astaxanthin applied in aquaculture, the green unicellular freshwater alga *Haematococcus* 

**Figure 4.** Figure 4. Chemical structure of β-carotene and of the xanthophylls astaxanthin and lutein,

β-Carotene is one of the important members of the family of carotenoids; a group of natural fat-soluble stereoisometric pigments. β-Carotene shows pro-vitamin A activity and as such it plays an important role in the human body[25]. β-Carotene can be also used as a coloring agent. Therefore, β-carotene has several applications in food, pharmaceuticals and cosmetics. The great demand of β-carotene has been met by industry, mainly by synthetic production. Increasing demand for natural carotenoids has resulted in growing interest in extracting β-carotene from different natural sources. *Dunaliella salina* is the main source for the natural β-carotene in the market. The estimated market size for natural β-carotene is 10- 100 tonnes.year-1 and its price is >750 €.Kg-1 [26]. In addition, β-carotene (like other carotenoids) is a strong antioxidant, scavenging potentially harmful oxy radicals, which are commonly associated with the induction of certain cancers (Leach et al., 1998) and there is an inverse relation between the consumption of certain carotenoids and the risk of cancer [25]. The demonstrated antioxidant activity of carotenoids is the basis of the protective action of these compounds against oxidative stress in many organisms and situations. Effects of carotenoids on human health are, in general, associated with their antioxidant properties. Notwithstanding, not all of the biological activities ascribed to carotenoids must be necessarily linked to their ability to prevent accumulation of free radicals and reactive oxygen species. The halophilic green biflagellate microalga *Dunaliella salina* has since long been recognized as an efficient biological source of this carotenoid. Many epidemiological studies suggest that humans fed on a diet high in β-carotene from *Dunaliella*, which maintains higher than average levels of serum carotenoids, have a lower incidence of several types of cancer and degenerative diseases [27]. The xanthophyll astaxanthin has many applications in nutraceuticals, cosmetics, and food and feed industries. Recently, a variety of

*pluvialis* has been explored by biotechnology companies [24].

main carotenoids from microalgae with commercial interest

**Figure 3.** Some sterols found in marine and freshwater microalgae (modified from Ponomarenko et al. [34]

#### **5.3. Pigmentation in aquacultures**

Astaxanthin (Figure 4) is a red pigment common to several aquatic organisms including microalgae, seagrasses, shrimp, lobsters and fish such as salmon and trout. Crustaceans are unable to synthesize carotenoids de novo and require astaxanthin (or appropriate precursors) in their diet in order to acquire the adequate color for seafoodmarket acceptance [23]. Several natural sources–such as the algae *Dunaliella salina* and *Spirulina maxima*–or synthetic β-carotene, canthaxanthin and astaxanthin have been used for this purpose. Astaxanthin is, in fact, one of the most expensive components of salmon farming, accounting for about 15%of total production costs [48]. Among the several natural sources of astaxanthin applied in aquaculture, the green unicellular freshwater alga *Haematococcus pluvialis* has been explored by biotechnology companies [24].

280 Plant Science

groups of algae [22].

[34]

**5.3. Pigmentation in aquacultures** 

matter input into an aquatic environment. Furthermore, sterols have a relatively high resistance to degradation when settled in anoxic sediments and persist in the environment for a longer period of time. Of all the sterol compounds, cholesterol is the most abundant and ubiquitous one in the environment, which is due to it having a variety of sources [21]. Most biologically produced sterols are planar 3-hydroxy tetracyclic structures commonly containing a methyl- or ethyl- substituted C7-C11 hydrocarbon side chain, and exhibiting a range of methyl-substitution (C4, C14) patterns on the polycyclic nucleus with varying degrees and positions of unsaturation (C5, C7, C8). The rigid structure of the sterols (Figure 3), caused by the fused ring system, provides the cell membrane integrity and stability thus, holds the membrane together. In general, there is not a specific sterol that can be uniquely linked to one algal source. Many of the sterols previously discussed are also found in other

**Figure 3.** Some sterols found in marine and freshwater microalgae (modified from Ponomarenko et al.

Astaxanthin (Figure 4) is a red pigment common to several aquatic organisms including microalgae, seagrasses, shrimp, lobsters and fish such as salmon and trout. Crustaceans are unable to synthesize carotenoids de novo and require astaxanthin (or appropriate

**Figure 4.** Figure 4. Chemical structure of β-carotene and of the xanthophylls astaxanthin and lutein, main carotenoids from microalgae with commercial interest

β-Carotene is one of the important members of the family of carotenoids; a group of natural fat-soluble stereoisometric pigments. β-Carotene shows pro-vitamin A activity and as such it plays an important role in the human body[25]. β-Carotene can be also used as a coloring agent. Therefore, β-carotene has several applications in food, pharmaceuticals and cosmetics. The great demand of β-carotene has been met by industry, mainly by synthetic production. Increasing demand for natural carotenoids has resulted in growing interest in extracting β-carotene from different natural sources. *Dunaliella salina* is the main source for the natural β-carotene in the market. The estimated market size for natural β-carotene is 10- 100 tonnes.year-1 and its price is >750 €.Kg-1 [26]. In addition, β-carotene (like other carotenoids) is a strong antioxidant, scavenging potentially harmful oxy radicals, which are commonly associated with the induction of certain cancers (Leach et al., 1998) and there is an inverse relation between the consumption of certain carotenoids and the risk of cancer [25]. The demonstrated antioxidant activity of carotenoids is the basis of the protective action of these compounds against oxidative stress in many organisms and situations. Effects of carotenoids on human health are, in general, associated with their antioxidant properties. Notwithstanding, not all of the biological activities ascribed to carotenoids must be necessarily linked to their ability to prevent accumulation of free radicals and reactive oxygen species. The halophilic green biflagellate microalga *Dunaliella salina* has since long been recognized as an efficient biological source of this carotenoid. Many epidemiological studies suggest that humans fed on a diet high in β-carotene from *Dunaliella*, which maintains higher than average levels of serum carotenoids, have a lower incidence of several types of cancer and degenerative diseases [27]. The xanthophyll astaxanthin has many applications in nutraceuticals, cosmetics, and food and feed industries. Recently, a variety of additional potential applications of this carotenoid, mainly related to human health and nutrition properties, have been claimed [28]. Lutein is among the most important carotenoids in foods and human serum and, together with zeaxanthin, is the essential component of the pigment present in the macula lutea (or yellow spot) in the eye retina and in the eye lens. Lutein is used as food dyes and especially as feed additives in aquaculture and poultry farming. During the last few years, additional applications for lutein have received considerable interest, especially those related to human health. Mainly on the basis of epidemiological studies, lutein is currently considered as effective agent for the prevention of a variety of human diseases. The microalga *Muriellopsis* sp. and other chlorophycean species are able to accumulate lutein as a part of their biomass. An established commercial system for the production of lutein from microalgae does not exist yet, although the basis for outdoor production of lutein-rich cells of strains of Muriellopsis and *Scenedesmus* at a pilot scale has alreadybeen set up [29].

Microalgal Biotechnology: Prospects and Applications 283

involve with the oxidation process by scavenging free radicals, chelating catalytic metals and by acting as oxygen scavengers [33]. Recently many researchers are interested in finding any natural antioxidants having safety and effectiveness, which can be substituted for current and commercial synthetic antioxidants, BHA and BHT. Microalgae have become good candidates for sources of natural antioxidants, as revealed by a number of recent studies [34-35]. Algae contain several enzymatic and nonenzymatic antioxidant defense systems to maintain the concentration of ROS (O2- and H2O2) to protect cells from damage [36]. The main cellular components susceptible to damage by these ROS are lipids (peroxidation of poly-unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids. The essential for ROS detoxification during normal metabolism and particularly during stress, are antioxidant defenses system [37]. The primary scavenging enzymatic defenses system include SOD, calalase (CAT) and glutathione peroxidase, (GPX) and peroxiredoxin (PrxR) [38]. These enzymic detoxification system involving the action of SOD and reductase, either quench toxic compounds or regenerate antioxidants with the help of reducing power provided by photosynthesis [39]. However, at low levels, H2O2 resulted in induction of defense genes such as glutathione Stransferase and glutathione peroxidase. The hydrophilic antioxidants AA and GSH effectively scavenge oxygen radicals. Carotenoids and TOH remove ROS directly from the pigment bed [40]. Also, Foyer and Noctor [41] reported that the changes in ROS, fluctuations in the antioxidants concentrations in photosynthetic cells might have important consequences not only for defense metabolism but also for the regulation of genes associated with adaptive responses. Several bioactive metabolites produced by cyanobacteria and algae have been discovered by screening programs, employing target organisms quite unrelated to those for which the metabolites evolved [42]. Shanab et al. [43] studied the antioxidant activity of aqueous extracts of nine microalgal species namely, *Nostoc muscorum, Anabaena flos aquae, Anabaena oryza*e, *Nostoc humifusum*, *Oscillatoria* sp., *Spirulina platensis, Phormedium fragile*, *Wollea saccata* and *Chlorella vulgaris*. Antioxidant activity of the algal extracts was performed using 2,2 diphenyl-1-picrylhydrazyl (DPPH) test


revealed higher antioxidant activity than DPPH mehod. Concerning DPPH, the antioxidant activity of nine tested algal species ranged between 30.1 and 72.4% comparing with the standarad antioxidant BHT (80.2%). Using ABTS method, which was more sensitvie than the DPPH method (Figure 5), the antioxidant activity ranged between 31.2 and 75.9% (Standarad BHT showed 85.6%); *Spirulina platensis*, *Oscillatoria* sp, *Anabaena flous-aqua* and *Nostoc muscorum* recorded the highest (75.9, 75.6, 73.6 and 72.8%, respectively) antioxidant activity which is could be attributed to the extracellular and intracellular secondary metabolits content (Total phenolic content, terpenoids and alkaloids) of these microalgae (Tables 1,2). The extractracellular phytochemicals metabolites (%) released in the algal cultures show large variability. *Anabaena oryza*, *Phomidium fragile* and *Wallea saccate* (Table 1) recorded the highest extracellular total phenolic compounds (0.0085, 0.0078 and 0.0074% respectively). The highest terpenoids contents were achieved by *Phormidium fragile*, *Spirulina platensis* and *Wollea saccata* (0.0055. 0.0050 and 0.0049%, respectively). The maximum values of the extracellular alkaloids were recorded by *Anabaena oryza*, *Phomidium fragile*, *Anabaena oryza*, *Spirulina platensis* and *Phomidium fragile* (0.075, 0.068 and 0.068%, respectively). While,

and 2,2/

#### **5.4. Mycosporine-like amino acids**

A remarkable group of marine natural products are the mycosporine-like amino acids (MAAs). An outstanding characteristic of these compounds is their high UV absorption with molar absorptivities (ε) of around 40 000 l mol-1 cm-1 (e.g. Takano et al. [30]). MAAs are water-soluble, low molecular-weight (generally <400) compounds composed of either an aminocyclohexenone or an aminocyclohexenimine ring, carrying nitrogen or amino alcohol substituents [31]. They are found in a wide variety of marine, freshwater and to a smaller degree in terrestrial organisms. There is limited evidence that MAAs are derived from early steps of the shikimate pathway. However, the biochemical pathway of MAA synthesis is still largely unknown, as well as its genetic base. The most primitive organisms capable of MAA synthesis are cyanobacteria [32].

## **6. Biological activity of microalgae**

Many of the microalgal metabolites have chemical structure and possess interesting biological activity. Microalgae are a unique source of therapeutic substances, particularly from cyanobacteria. Among cyanobacteria *Spirulina* sp. has undergone numerous and rigorous toxicological studies that have highlighted its potential therapeutic applications in the area of immunomodulation, anticancer, antiviral, and cholesterol reduction effects.

#### **6.1. Antioxidant activity**

Hydrogen peroxide is a product of microalgae and plants through of photosynthesis, photorespiration, respiration and other metabolic processes, as result from the enzymatic activity of glycolate oxidase, urate oxidase and amino acid oxidase. However, major pathway for production of H2O2 is conversion from superoxide (O2-) produced through the transfer of an electron from ferredoxin of photosystem I (PSI) to O2 (Mehler reaction) by the action of Superoxide Dismutase (SOD). However, it is suspected that those antioxidants are responsible for some side effects such as liver damage and carcinogenesis. Antioxidants can involve with the oxidation process by scavenging free radicals, chelating catalytic metals and by acting as oxygen scavengers [33]. Recently many researchers are interested in finding any natural antioxidants having safety and effectiveness, which can be substituted for current and commercial synthetic antioxidants, BHA and BHT. Microalgae have become good candidates for sources of natural antioxidants, as revealed by a number of recent studies [34-35]. Algae contain several enzymatic and nonenzymatic antioxidant defense systems to maintain the concentration of ROS (O2- and H2O2) to protect cells from damage [36]. The main cellular components susceptible to damage by these ROS are lipids (peroxidation of poly-unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids. The essential for ROS detoxification during normal metabolism and particularly during stress, are antioxidant defenses system [37]. The primary scavenging enzymatic defenses system include SOD, calalase (CAT) and glutathione peroxidase, (GPX) and peroxiredoxin (PrxR) [38]. These enzymic detoxification system involving the action of SOD and reductase, either quench toxic compounds or regenerate antioxidants with the help of reducing power provided by photosynthesis [39]. However, at low levels, H2O2 resulted in induction of defense genes such as glutathione Stransferase and glutathione peroxidase. The hydrophilic antioxidants AA and GSH effectively scavenge oxygen radicals. Carotenoids and TOH remove ROS directly from the pigment bed [40]. Also, Foyer and Noctor [41] reported that the changes in ROS, fluctuations in the antioxidants concentrations in photosynthetic cells might have important consequences not only for defense metabolism but also for the regulation of genes associated with adaptive responses. Several bioactive metabolites produced by cyanobacteria and algae have been discovered by screening programs, employing target organisms quite unrelated to those for which the metabolites evolved [42]. Shanab et al. [43] studied the antioxidant activity of aqueous extracts of nine microalgal species namely, *Nostoc muscorum, Anabaena flos aquae, Anabaena oryza*e, *Nostoc humifusum*, *Oscillatoria* sp., *Spirulina platensis, Phormedium fragile*, *Wollea saccata* and *Chlorella vulgaris*. Antioxidant activity of the algal extracts was performed using 2,2 diphenyl-1-picrylhydrazyl (DPPH) test and 2,2/ -azino-bis(ethylbenzthiazoline-6-sulfonic acid (ABTS) radical action assay which revealed higher antioxidant activity than DPPH mehod. Concerning DPPH, the antioxidant activity of nine tested algal species ranged between 30.1 and 72.4% comparing with the standarad antioxidant BHT (80.2%). Using ABTS method, which was more sensitvie than the DPPH method (Figure 5), the antioxidant activity ranged between 31.2 and 75.9% (Standarad BHT showed 85.6%); *Spirulina platensis*, *Oscillatoria* sp, *Anabaena flous-aqua* and *Nostoc muscorum* recorded the highest (75.9, 75.6, 73.6 and 72.8%, respectively) antioxidant activity which is could be attributed to the extracellular and intracellular secondary metabolits content (Total phenolic content, terpenoids and alkaloids) of these microalgae (Tables 1,2). The extractracellular phytochemicals metabolites (%) released in the algal cultures show large variability. *Anabaena oryza*, *Phomidium fragile* and *Wallea saccate* (Table 1) recorded the highest extracellular total phenolic compounds (0.0085, 0.0078 and 0.0074% respectively). The highest terpenoids contents were achieved by *Phormidium fragile*, *Spirulina platensis* and *Wollea saccata* (0.0055. 0.0050 and 0.0049%, respectively). The maximum values of the extracellular alkaloids were recorded by *Anabaena oryza*, *Phomidium fragile*, *Anabaena oryza*, *Spirulina platensis* and *Phomidium fragile* (0.075, 0.068 and 0.068%, respectively). While,

282 Plant Science

additional potential applications of this carotenoid, mainly related to human health and nutrition properties, have been claimed [28]. Lutein is among the most important carotenoids in foods and human serum and, together with zeaxanthin, is the essential component of the pigment present in the macula lutea (or yellow spot) in the eye retina and in the eye lens. Lutein is used as food dyes and especially as feed additives in aquaculture and poultry farming. During the last few years, additional applications for lutein have received considerable interest, especially those related to human health. Mainly on the basis of epidemiological studies, lutein is currently considered as effective agent for the prevention of a variety of human diseases. The microalga *Muriellopsis* sp. and other chlorophycean species are able to accumulate lutein as a part of their biomass. An established commercial system for the production of lutein from microalgae does not exist yet, although the basis for outdoor production of lutein-rich cells of strains of Muriellopsis

A remarkable group of marine natural products are the mycosporine-like amino acids (MAAs). An outstanding characteristic of these compounds is their high UV absorption with molar absorptivities (ε) of around 40 000 l mol-1 cm-1 (e.g. Takano et al. [30]). MAAs are water-soluble, low molecular-weight (generally <400) compounds composed of either an aminocyclohexenone or an aminocyclohexenimine ring, carrying nitrogen or amino alcohol substituents [31]. They are found in a wide variety of marine, freshwater and to a smaller degree in terrestrial organisms. There is limited evidence that MAAs are derived from early steps of the shikimate pathway. However, the biochemical pathway of MAA synthesis is still largely unknown, as well as its genetic base. The most primitive organisms capable of

Many of the microalgal metabolites have chemical structure and possess interesting biological activity. Microalgae are a unique source of therapeutic substances, particularly from cyanobacteria. Among cyanobacteria *Spirulina* sp. has undergone numerous and rigorous toxicological studies that have highlighted its potential therapeutic applications in the area of immunomodulation, anticancer, antiviral, and cholesterol reduction effects.

Hydrogen peroxide is a product of microalgae and plants through of photosynthesis, photorespiration, respiration and other metabolic processes, as result from the enzymatic activity of glycolate oxidase, urate oxidase and amino acid oxidase. However, major pathway for production of H2O2 is conversion from superoxide (O2-) produced through the transfer of an electron from ferredoxin of photosystem I (PSI) to O2 (Mehler reaction) by the action of Superoxide Dismutase (SOD). However, it is suspected that those antioxidants are responsible for some side effects such as liver damage and carcinogenesis. Antioxidants can

and *Scenedesmus* at a pilot scale has alreadybeen set up [29].

**5.4. Mycosporine-like amino acids** 

MAA synthesis are cyanobacteria [32].

**6.1. Antioxidant activity** 

**6. Biological activity of microalgae** 

the highest percentages of these metabolities complaining with the released extracellularly metabolities, *Spirulina platensis*, *Nostoc muscourum* and *Oscillatoria* sp. recorded the greatest intracellular total phenolic compounds (0.71, 0.6 and 0.55% respectively), while *Wollea saccata* showed the least content (0.1%) as shown in Table (2).

Microalgal Biotechnology: Prospects and Applications 285

**Table 3.** Phycobilins pigments (mg/ml) in different aqueous filtrate of some microalgae [43]

*Nostoc muscorum* contained the greatest allophycocyanin (APC) pigment (0.14 mg/l), while, *Oscillatoria* sp have the least content (0.001 mg/l). Concerning total phycobilin pigments (phycocyanin and allophycocyanin), *Nostoc muscrum*, *Phormedium fragile* recorded the highest contents (0.229 and 0.16 mg/l) followed by that of S. platensis (0.143 mg/ml), while *Anabaena oryzae* showed the least total phycobilin pigment content (0.011 mg/l).*Chlorella vulgaris* is green alga which have other pigments (Chlorophyll and carotenoids) than the phycobilins (absent). Aqueous extracts of the tested algal species showed wide range of colours (green, blue, violet, pink, ligh-blue) in spite of the fact that eight of the tested algae were of cyanobacteria and only one species was a green alga, their water extracts showed highly variable colors (Figure 6) which may be attributed in part to their phycobiliprotein constituents (ratios of phycocyanin to allophycocyanin and approximately absence of phycoerytherin pigments), and in part to the produced major polar secondary metabolites. All these substances may not only caused the alteration of the pH values of the algal aqueous extracts, but also the induced biological activities which may be attributed to the synergistic effects of these compounds. The aqueous extract of the tested algal species (8 cyanobacteria and one green alga) have variable colors (Figure 6) ranging from green, violet, blue, light blue and pink color, which can be used as an additive coloring agents to different food products (natural, non toxic) instead of the synthetic coloring substances which may be carcinogenic [43].

**Figure 6.** Color of aqueous extracts of the different microalgae (1. Nostoc muscorum, 2. Anabaena flos aquae, 3.Chlorella vulgari, 4. Oscillatoria sp., 5. Spirulina platensis, 6. Anabaena oryzae, 7. Wollea

saccat, 8. Nostoc humifusum and 9. Phormedium fragile [43]

Concerning terpenoids, *Anabaena flos aquae*, *Spirulina platensis* and *Wollea saccata*, recorded the highest contents (0.15, 0.14 and 0.14% respectively). Alkaloids determination in algal cultures showed that *S. platensis*, *Oscillatoria* sp. and *Chlorella vulgaris* showed the highest contents (3.02, 2.6 and 2.45% respectively). Phycobiliprotein pigments (Table 3) were determined in water extracts of the tested algal species. Normally, phycobilin pigments in cyanobacteria comprised phycocyanin, allophycocyanin and phycoerytherin (the blue, gray and red colors respectively). *Phormidium fragile* recorded the higher C-phycocyanin (CPC) content (0.13 mg/ml) while *Anabaena oryzae* and *A. flos aquae* recorded the least and absence of phycocyanin (0.0089 and 0.0 mg/l respectively).

**Figure 5.** Antioxidant activity of algal wter extracts at 100 g/ml against DPPH (a) and ABTS (b) radicals [43]


**Table 1.** Secondary metabolites (as mg/100 g) in algal filterates (extracellular) [43]


**Table 2.** Secondary metabolites (as %) in algal cells (inracellular) [43]



**Table 3.** Phycobilins pigments (mg/ml) in different aqueous filtrate of some microalgae [43]

284 Plant Science

[43]

the highest percentages of these metabolities complaining with the released extracellularly metabolities, *Spirulina platensis*, *Nostoc muscourum* and *Oscillatoria* sp. recorded the greatest intracellular total phenolic compounds (0.71, 0.6 and 0.55% respectively), while *Wollea* 

Concerning terpenoids, *Anabaena flos aquae*, *Spirulina platensis* and *Wollea saccata*, recorded the highest contents (0.15, 0.14 and 0.14% respectively). Alkaloids determination in algal cultures showed that *S. platensis*, *Oscillatoria* sp. and *Chlorella vulgaris* showed the highest contents (3.02, 2.6 and 2.45% respectively). Phycobiliprotein pigments (Table 3) were determined in water extracts of the tested algal species. Normally, phycobilin pigments in cyanobacteria comprised phycocyanin, allophycocyanin and phycoerytherin (the blue, gray and red colors respectively). *Phormidium fragile* recorded the higher C-phycocyanin (CPC) content (0.13 mg/ml) while *Anabaena oryzae* and *A. flos aquae* recorded the least and absence

**Figure 5.** Antioxidant activity of algal wter extracts at 100 g/ml against DPPH (a) and ABTS (b) radicals

**Table 1.** Secondary metabolites (as mg/100 g) in algal filterates (extracellular) [43]

**Table 2.** Secondary metabolites (as %) in algal cells (inracellular) [43]

*saccata* showed the least content (0.1%) as shown in Table (2).

of phycocyanin (0.0089 and 0.0 mg/l respectively).

*Nostoc muscorum* contained the greatest allophycocyanin (APC) pigment (0.14 mg/l), while, *Oscillatoria* sp have the least content (0.001 mg/l). Concerning total phycobilin pigments (phycocyanin and allophycocyanin), *Nostoc muscrum*, *Phormedium fragile* recorded the highest contents (0.229 and 0.16 mg/l) followed by that of S. platensis (0.143 mg/ml), while *Anabaena oryzae* showed the least total phycobilin pigment content (0.011 mg/l).*Chlorella vulgaris* is green alga which have other pigments (Chlorophyll and carotenoids) than the phycobilins (absent).

Aqueous extracts of the tested algal species showed wide range of colours (green, blue, violet, pink, ligh-blue) in spite of the fact that eight of the tested algae were of cyanobacteria and only one species was a green alga, their water extracts showed highly variable colors (Figure 6) which may be attributed in part to their phycobiliprotein constituents (ratios of phycocyanin to allophycocyanin and approximately absence of phycoerytherin pigments), and in part to the produced major polar secondary metabolites. All these substances may not only caused the alteration of the pH values of the algal aqueous extracts, but also the induced biological activities which may be attributed to the synergistic effects of these compounds. The aqueous extract of the tested algal species (8 cyanobacteria and one green alga) have variable colors (Figure 6) ranging from green, violet, blue, light blue and pink color, which can be used as an additive coloring agents to different food products (natural, non toxic) instead of the synthetic coloring substances which may be carcinogenic [43].

**Figure 6.** Color of aqueous extracts of the different microalgae (1. Nostoc muscorum, 2. Anabaena flos aquae, 3.Chlorella vulgari, 4. Oscillatoria sp., 5. Spirulina platensis, 6. Anabaena oryzae, 7. Wollea saccat, 8. Nostoc humifusum and 9. Phormedium fragile [43]

Phycobiliprotein pigments were known by its antioxidant activity [44], increasing of these pigments production as a result of doubling nitrate concentration in the growth culture media, led to a progressive increase in the antioxidant activity recorded by both DPPH and ABTS assays in the two cyanobacteria under investigation.Keeping in mind that, synergetic effect occurred between the polar secondry metabolites especially the phenolic compounds and the polysaccharides in antioxidant activity. Increasing nitrate concentrations in the culture media of both cyanobacteria species (*N. muscorum* and *Oscillatoria* sp) led to a marked enhancement in phycobiliprotein production (Table 4) which was translated in an obvious increase in antioxidant activity (by DPPH and ABTS) in both species under study while decreasing the nitrate content, phycobilin pigments production were consequently decreased in both species and its complete absence was recorded on nitrogen starvation especially in case of *Oscillatoria* sp., and no allophycocyanin pigments were produced as found by Shanab et al.[43]. They also investigated that increasing nitrate conc. and the consequent increase in phycobilin pigments production, have the major role in enhancing the antioxidant activity may be attributed. The decrease in nitrate conc. was followed by an obvious decrease in phycobilin pigment and even an absence of one of its constituents on nitrate starvation. The antioxidant activity in both species (by both assays) was apparently not affected comparing with the control (1.5 g/l nitrate). Under stress conditions, it was known that, deviation in metabolic pathways may occur. In presence of nitrate, nitrogenous compounds, including the phycobilin pigments were increasingly produced leading, together with other antioxidant active secondary metabolites (as phenolics), to a marked increase in biological activity.

Microalgal Biotechnology: Prospects and Applications 287

(Comparable to those in presence of high nitrate content (6-9 g/l) was largely due to the high production of the carbon skeleton compounds (phenolic compounds) which show potent antioxidant activity [45]. Shalaby et al. [46] stated that cultivation of *Spirulina platensis* under salt stress conditions (0.02 M as control), 0.04 and 0.08 M NaCl led to a remarkable alteration of algal metabolism as well as an enhancement or induction of biologically active compounds. Biochemical analysis of salt stressed algal revealed that lipid content was slightly increased together with certain saturated and unsaturated fatty acids especially the

Today cancer is the largest single cause of death in men and women, and chemoprevention has been a promising anticancer approach aimed at reducing themorbidity andmortality of cancer by delaying the process of carcinogenesis. A variety of compounds fromnature sources have been shown to be beneficial for the inhibition of cancer, such as flavonoids, phenolic acids, carotenoids, etc.; the mechanisms which suppress tumorgenesis often involve inhibition of tumor cell mediated protease activity, attenuation of tumor angiogenesis, promotion of cell cycle arrest, induction of apoptosis and immunostimulation, etc. In addition, Chinery et al. [47] also reported their use with the chemotherapy agents 5 fluorouracil and antioxidants could cause complete remissions in colorectal cancer, where only partial remission is possiblewith chemotherapy agents only; therefore, antioxidants have been proposed to have potential for the prevention and treatment of diseases associated with active oxygen species, especially in cancer diseases. Moreover, experimental and epidemiological evidence suggests that anti-inflammatory drugs may also decrease the incidence of mammary cancer, tumor burden, and tumor volume [48].The medicinal value of cyanobacteria was appreciated as early as 1500 Bc, when strains of Nostoc were used to treat gout, fistula and several forms of cancer. Cyanobacteria are a rich source of potentially useful natural products. Over 40 different Nostocales species, the majority of which are Anabaena and Nostoc spp. Produce over 120 natural products (Secondry metabolities) having activities such as anti-HIV anticancer, antifungal, antimalarial and antimicrobial. Cyanovirin (CV-N, cyanoviorin-N), a 101 amino acid protein extracted from *Nostoc ellipsosporum* was found to have potent activity against all human immunodeficiency viruses such as HIV-1, M and T tropic strains of HIV-1, HIV-2, SIV (Simian), and FIV (Feline) [7]. The cosmopolitan distribution of cyanobacteria indicates that they can cope with a wide spectrum of global environmental stress, such as heat, cold, desiccation, salinity, nitrogen starvation, photooxidation, anaerobiosis and osmotic stress. They have developed a number of mechanisms by which cyanobacteria defend themselves against environmental stressors. Important among them are the production of photoprotective compounds such as mycosporine-like amino acids (MAAs) and Scytonemin enzymes such as superoxide dismutase, catalase and peroxidases repaire of DNA damage and synthesis of shock proteins [49]. Shanab et al. [43] investigated anticancer efficiency of the algal water extracts against Ehrlich Ascites Carcinoma cell (EACC) and Human hepatocellular cancer cell line (HepG2). Anticancer efficiency of the algal water extracts was investigated against Ehrlich

polyunsaturated ones (γ-inolenic acid, omega 3 fatty acid).

**6.2. Anticancer activity** 


**Table 4.** Antioxidant activity of the nitrogen stressed promising algal species using DPPH and ABTS radicals [43]

The decrease in nitrate content induced a stress condition and not only a decrease in nitrogen skeleton compounds as phycobilin pigment production, but an increase in the carbon skeleton compounds (as phenolics) as a result of metabolic alterations under these stress conditions. So on decreasing nitrate content, the antioxidant activity remain at a level comparable or even higher than the control due to the synergistic effect of the phycobilin pigment and the phenolic compounds produced in excess under stress nitrate condition which have high redox potentials. On nitrogen starvation the recorded antioxidant activity (Comparable to those in presence of high nitrate content (6-9 g/l) was largely due to the high production of the carbon skeleton compounds (phenolic compounds) which show potent antioxidant activity [45]. Shalaby et al. [46] stated that cultivation of *Spirulina platensis* under salt stress conditions (0.02 M as control), 0.04 and 0.08 M NaCl led to a remarkable alteration of algal metabolism as well as an enhancement or induction of biologically active compounds. Biochemical analysis of salt stressed algal revealed that lipid content was slightly increased together with certain saturated and unsaturated fatty acids especially the polyunsaturated ones (γ-inolenic acid, omega 3 fatty acid).

#### **6.2. Anticancer activity**

286 Plant Science

increase in biological activity.

radicals [43]

Phycobiliprotein pigments were known by its antioxidant activity [44], increasing of these pigments production as a result of doubling nitrate concentration in the growth culture media, led to a progressive increase in the antioxidant activity recorded by both DPPH and ABTS assays in the two cyanobacteria under investigation.Keeping in mind that, synergetic effect occurred between the polar secondry metabolites especially the phenolic compounds and the polysaccharides in antioxidant activity. Increasing nitrate concentrations in the culture media of both cyanobacteria species (*N. muscorum* and *Oscillatoria* sp) led to a marked enhancement in phycobiliprotein production (Table 4) which was translated in an obvious increase in antioxidant activity (by DPPH and ABTS) in both species under study while decreasing the nitrate content, phycobilin pigments production were consequently decreased in both species and its complete absence was recorded on nitrogen starvation especially in case of *Oscillatoria* sp., and no allophycocyanin pigments were produced as found by Shanab et al.[43]. They also investigated that increasing nitrate conc. and the consequent increase in phycobilin pigments production, have the major role in enhancing the antioxidant activity may be attributed. The decrease in nitrate conc. was followed by an obvious decrease in phycobilin pigment and even an absence of one of its constituents on nitrate starvation. The antioxidant activity in both species (by both assays) was apparently not affected comparing with the control (1.5 g/l nitrate). Under stress conditions, it was known that, deviation in metabolic pathways may occur. In presence of nitrate, nitrogenous compounds, including the phycobilin pigments were increasingly produced leading, together with other antioxidant active secondary metabolites (as phenolics), to a marked

**Table 4.** Antioxidant activity of the nitrogen stressed promising algal species using DPPH and ABTS

The decrease in nitrate content induced a stress condition and not only a decrease in nitrogen skeleton compounds as phycobilin pigment production, but an increase in the carbon skeleton compounds (as phenolics) as a result of metabolic alterations under these stress conditions. So on decreasing nitrate content, the antioxidant activity remain at a level comparable or even higher than the control due to the synergistic effect of the phycobilin pigment and the phenolic compounds produced in excess under stress nitrate condition which have high redox potentials. On nitrogen starvation the recorded antioxidant activity Today cancer is the largest single cause of death in men and women, and chemoprevention has been a promising anticancer approach aimed at reducing themorbidity andmortality of cancer by delaying the process of carcinogenesis. A variety of compounds fromnature sources have been shown to be beneficial for the inhibition of cancer, such as flavonoids, phenolic acids, carotenoids, etc.; the mechanisms which suppress tumorgenesis often involve inhibition of tumor cell mediated protease activity, attenuation of tumor angiogenesis, promotion of cell cycle arrest, induction of apoptosis and immunostimulation, etc. In addition, Chinery et al. [47] also reported their use with the chemotherapy agents 5 fluorouracil and antioxidants could cause complete remissions in colorectal cancer, where only partial remission is possiblewith chemotherapy agents only; therefore, antioxidants have been proposed to have potential for the prevention and treatment of diseases associated with active oxygen species, especially in cancer diseases. Moreover, experimental and epidemiological evidence suggests that anti-inflammatory drugs may also decrease the incidence of mammary cancer, tumor burden, and tumor volume [48].The medicinal value of cyanobacteria was appreciated as early as 1500 Bc, when strains of Nostoc were used to treat gout, fistula and several forms of cancer. Cyanobacteria are a rich source of potentially useful natural products. Over 40 different Nostocales species, the majority of which are Anabaena and Nostoc spp. Produce over 120 natural products (Secondry metabolities) having activities such as anti-HIV anticancer, antifungal, antimalarial and antimicrobial. Cyanovirin (CV-N, cyanoviorin-N), a 101 amino acid protein extracted from *Nostoc ellipsosporum* was found to have potent activity against all human immunodeficiency viruses such as HIV-1, M and T tropic strains of HIV-1, HIV-2, SIV (Simian), and FIV (Feline) [7]. The cosmopolitan distribution of cyanobacteria indicates that they can cope with a wide spectrum of global environmental stress, such as heat, cold, desiccation, salinity, nitrogen starvation, photooxidation, anaerobiosis and osmotic stress. They have developed a number of mechanisms by which cyanobacteria defend themselves against environmental stressors. Important among them are the production of photoprotective compounds such as mycosporine-like amino acids (MAAs) and Scytonemin enzymes such as superoxide dismutase, catalase and peroxidases repaire of DNA damage and synthesis of shock proteins [49]. Shanab et al. [43] investigated anticancer efficiency of the algal water extracts against Ehrlich Ascites Carcinoma cell (EACC) and Human hepatocellular cancer cell line (HepG2). Anticancer efficiency of the algal water extracts was investigated against Ehrlich Ascites Carcinoma cell (EACC) and Human hepatocellular cancer cell line (HepG2). The anticancer efficiency of the algal aqueous extracts illustrated in Figure (7) and using EACC and HepG2 cell lines, recorded that the anticancer activity ranged between 15.68 to 87.25 % in case of EACC cell line and from 9.5 to 89.4% using HepG2 cell line *Nostoc muscorum*  aqueous extracts recorded the highest anticancer activity in both cell lines (87.25% in case of EACC and 89.4% in case of HepG2), followed by *Oscillatoria* sp. (67.40 and 77.8% in EACC and HepG2 respectively). In case of *N. muscorum*, the anticancer activity against EACC cell line ranged between 83.0 and 90.4% at all nitrate concentrations (increase and decrease) compared to the control (85.9%). Comparable anticancer activity was recorded at both the highest nitrate conc and starvation (90.4 and 89.9% respectively). The anticancer activity against HepG2 cell line recorded more or less comparable activities were recorded at most nitrate conc compared to the control (85.6, 86.9, 88.7 and 88.6 % at 3, 6, 9 and 1.5 g/l). At nitrate starvation the highest anticancer activity against HepG2 cell line was recorded (92.3%). In case of *Oscillatoria* sp., the anticancer activity against EACC and HepG2 recorded an increase in activity on both increasing and decreasing nitrate conc comparing with the control. Higher activity was recorded against both cell lines at higher nitrate conc (82.6 and 75.9% in case of EACC and HepG2 respectively) and at nitrate starvation (82.9 and 82.0% respectively) compared to the control (68.3 and 70.4 % against EACC and HepG2 respectively). Water extracts of the tested promising algal species demonstrated higher anticancer efficiencies against both EACC and HepG2 cell lines (87.25 and 89.4% respectively) in case of *N. muscorum* and 67.40 and 77.8% in case of *Oscillatoria* sp.). Under stress nitrogen conditions, these two cyanobacteria species recorded higher anticancer activities on exess limitation or starvation of nitrate comparing with its normal content in growth media.

Microalgal Biotechnology: Prospects and Applications 289

The recorded maximum activity in both species against both cell lines at the highest nitrate content (9 g/L) may be attributed mainly to the higher content of the phycobiliprotein pigments produced under excess nitrate contents. Nitrate limitation and starvation, in spite of the caused decrease in phycobilin pigment production due to metabolic alteration expected under stress conditions, the carbon skeleton compounds as phenolic may replace phycobilin shortage in inducing similar anticancer activity of or even higher efficiency caused by great phycobilin contents at higher nitrate supplementation [43]. These results demonstrated that the compounds responsible for anticarcinogenic activity was highly polar as the phycobilins, phenolic compounds and polysaccharides which induced apoptosis of the cancer cells as reported by Aboul-Enein et al [50], which go parallel with these results coincides with the results obtained by Wang et al [51] who reported that the aqueous extract of red algae mainly contain c-phycocyanin, exhibited higher antipraleferation inducing apoptosis body formation. The authors explained that phycocyanin interact with membrane associated B-tubulin and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), caused polymerization of microtubules and actins filaments leading to arrested the cell cycle at G0/G1 phase. As these aqueous extracts exhibited antioxidant and anticancer activities, its effect as coloring agent is amplified by these biological efficiencies which are very important for human health. Also, it can be used for the manufacture of pharmaceutical drugs

Table (5) recorded the anticancer efficiency of nitrate stressed *N. muscorum* and *Oscillatoria* sp. against EACC and HepG2 cell lines. The promising cyanobacterial species *N. muscorum* and *Oscillatoria* sp. induced both the highest antioxidant (by DPPH and ABTS methods) and anticancer activities (using EACC and HepG2 cell lines) which may be attributed to their large content in total phycobiliprotein pigments together with the higher secondary

The antimicrobial activity of microalgae has been attributed to compounds belonging to several chemical classes –including indoles, terpenes, acetogenins, phenols, fatty acids and volatile halogenated hydrocarbons [52] for instance, the antimicrobial activity of supercritical extracts obtained from the microalga *Chaetoceros muelleri* were related to its lipid composition [53]. However, the antimicrobial activity detected in several pressurized extracts from *Dunaliella salina* may be explained not only by several fatty acids, but also by such compounds as - and -ionone, -cyclocitral, neophytadiene and phytol. Efforts to identify the compounds directly responsible for those antimicrobial features –e.g. chlorellin [54] have been on the run, but are still relatively incipient owing the some new classes of compounds found. Microalgal cell-free extracts are already being tested as additives for food and feed formulation, in attempts to replace antimicrobial compounds of synthetic origin currently in use – including subtherapeutical doses of antibiotics employed as prophilatic measure in animal breeding [55]. Recall, in this regard, the growing resistance of some bacterial strains arising from the widespread and essentially unrestricted use of antibiotics in cattle handling, and by domestic

metabolites content (phenolic compounds, terpenoids, alkaloids [43].

(antioxidant and anticancer).

**6.3. Antimicrobial activity** 

**Figure 7.** (a, b). Anticancer activity of algal water extracts at 100 µg/ ml against EACC (a) and HepG2 (b) cell lines [43]

The recorded maximum activity in both species against both cell lines at the highest nitrate content (9 g/L) may be attributed mainly to the higher content of the phycobiliprotein pigments produced under excess nitrate contents. Nitrate limitation and starvation, in spite of the caused decrease in phycobilin pigment production due to metabolic alteration expected under stress conditions, the carbon skeleton compounds as phenolic may replace phycobilin shortage in inducing similar anticancer activity of or even higher efficiency caused by great phycobilin contents at higher nitrate supplementation [43]. These results demonstrated that the compounds responsible for anticarcinogenic activity was highly polar as the phycobilins, phenolic compounds and polysaccharides which induced apoptosis of the cancer cells as reported by Aboul-Enein et al [50], which go parallel with these results coincides with the results obtained by Wang et al [51] who reported that the aqueous extract of red algae mainly contain c-phycocyanin, exhibited higher antipraleferation inducing apoptosis body formation. The authors explained that phycocyanin interact with membrane associated B-tubulin and glyceraldehydes-3-phosphate dehydrogenase (GAPDH), caused polymerization of microtubules and actins filaments leading to arrested the cell cycle at G0/G1 phase. As these aqueous extracts exhibited antioxidant and anticancer activities, its effect as coloring agent is amplified by these biological efficiencies which are very important for human health. Also, it can be used for the manufacture of pharmaceutical drugs (antioxidant and anticancer).

Table (5) recorded the anticancer efficiency of nitrate stressed *N. muscorum* and *Oscillatoria* sp. against EACC and HepG2 cell lines. The promising cyanobacterial species *N. muscorum* and *Oscillatoria* sp. induced both the highest antioxidant (by DPPH and ABTS methods) and anticancer activities (using EACC and HepG2 cell lines) which may be attributed to their large content in total phycobiliprotein pigments together with the higher secondary metabolites content (phenolic compounds, terpenoids, alkaloids [43].

#### **6.3. Antimicrobial activity**

288 Plant Science

growth media.

(b) cell lines [43]

Ascites Carcinoma cell (EACC) and Human hepatocellular cancer cell line (HepG2). The anticancer efficiency of the algal aqueous extracts illustrated in Figure (7) and using EACC and HepG2 cell lines, recorded that the anticancer activity ranged between 15.68 to 87.25 % in case of EACC cell line and from 9.5 to 89.4% using HepG2 cell line *Nostoc muscorum*  aqueous extracts recorded the highest anticancer activity in both cell lines (87.25% in case of EACC and 89.4% in case of HepG2), followed by *Oscillatoria* sp. (67.40 and 77.8% in EACC and HepG2 respectively). In case of *N. muscorum*, the anticancer activity against EACC cell line ranged between 83.0 and 90.4% at all nitrate concentrations (increase and decrease) compared to the control (85.9%). Comparable anticancer activity was recorded at both the highest nitrate conc and starvation (90.4 and 89.9% respectively). The anticancer activity against HepG2 cell line recorded more or less comparable activities were recorded at most nitrate conc compared to the control (85.6, 86.9, 88.7 and 88.6 % at 3, 6, 9 and 1.5 g/l). At nitrate starvation the highest anticancer activity against HepG2 cell line was recorded (92.3%). In case of *Oscillatoria* sp., the anticancer activity against EACC and HepG2 recorded an increase in activity on both increasing and decreasing nitrate conc comparing with the control. Higher activity was recorded against both cell lines at higher nitrate conc (82.6 and 75.9% in case of EACC and HepG2 respectively) and at nitrate starvation (82.9 and 82.0% respectively) compared to the control (68.3 and 70.4 % against EACC and HepG2 respectively). Water extracts of the tested promising algal species demonstrated higher anticancer efficiencies against both EACC and HepG2 cell lines (87.25 and 89.4% respectively) in case of *N. muscorum* and 67.40 and 77.8% in case of *Oscillatoria* sp.). Under stress nitrogen conditions, these two cyanobacteria species recorded higher anticancer activities on exess limitation or starvation of nitrate comparing with its normal content in

**Figure 7.** (a, b). Anticancer activity of algal water extracts at 100 µg/ ml against EACC (a) and HepG2

The antimicrobial activity of microalgae has been attributed to compounds belonging to several chemical classes –including indoles, terpenes, acetogenins, phenols, fatty acids and volatile halogenated hydrocarbons [52] for instance, the antimicrobial activity of supercritical extracts obtained from the microalga *Chaetoceros muelleri* were related to its lipid composition [53]. However, the antimicrobial activity detected in several pressurized extracts from *Dunaliella salina* may be explained not only by several fatty acids, but also by such compounds as - and -ionone, -cyclocitral, neophytadiene and phytol. Efforts to identify the compounds directly responsible for those antimicrobial features –e.g. chlorellin [54] have been on the run, but are still relatively incipient owing the some new classes of compounds found. Microalgal cell-free extracts are already being tested as additives for food and feed formulation, in attempts to replace antimicrobial compounds of synthetic origin currently in use – including subtherapeutical doses of antibiotics employed as prophilatic measure in animal breeding [55].

Recall, in this regard, the growing resistance of some bacterial strains arising from the widespread and essentially unrestricted use of antibiotics in cattle handling, and by domestic


consumers use via self-prescription [56]. However, a key factor for their eventual economic feasibility is the possibility of operating large photobioreactors under aseptic conditions, which are able to produce biomass and metabolites to sufficiently high levels [57].

Microalgal Biotechnology: Prospects and Applications 291

occur in the Golgi apparatus [62]; these findings were confirmed in *Porphyridium* sp. [63] and other red microalgae [64]. More recently, [65] used 14C pulse-chase experiments and ultrastructural microscopy to conclude that brefeldin A–a membrane-traffic inhibitor of the Golgi apparatus, decreases the contents of the bound and the soluble forms of polysaccharides, while inhibiting cell-wall binding of polysaccharides to a greater extent than its soluble counterpart (in both actively growing and resting cells). Discovery of small molecules that can specifically disrupt a particular protein-protein interface remains a challenge-but is of a particular interest in virology, since the antiviral drugs currently

Most efforts were devoted to the study of antibiotic resistance in bacteria for several reasons: (i) bacterial infections are responsible for most community-acquired and nosocomial infections; (ii) the large and expanding number of antibacterial classes offers a more diverse range of resistance mechanisms; and (iii) the ability to move bacterial resistance determinants into standard, well-characterized bacterial strains facilitates more detailed studies of the underlying molecular mechanisms [66]. Pratt et al. [67] isolated the first antibacterial compound from a microalga, *Chlorella*; a mixture of fatty acids, viz. chlorellin, was found to be responsible for that inhibitory activity against both Gram+ and Grambacteria. Research aimed at identifying antibacterial active principles produced by microalgae has meanwhile boomed [68]. This realisation arose e.g. from the risk associated with several multidrug-resistant *Staphylococcus aureus* (MRSA) strains, which have been causing an increased concern in healthcare institutions worldwide – since they are not susceptible to most conventional antibiotics. Hence, discovery of novel antibacterial compounds following distinct biochemical mechanisms of action is urged. Antibiotics are typically less effective against Gram- bacteria because of their complex, multilayered cell wall structure – which makes it more difficult for the active compound to penetrate them [69]; this justifies why the antibacterial activity of the supernatant (and methanolic extracts) is more potent against Gram+ than Gram- bacteria [68,70]. The exact mechanism of action of fatty acids remains unknown: they may act upon multiple cellular targets, even though cell membranes are the most probable ones – as membrane damage will likely lead to cell leakage and reduction of nutrient uptake, besides inhibiting cellular respiration; conversely, Desbois [71] claimed a peroxidative process. Furthermore, compounds synthesized by *Scenedesmus costatum*, and partially purified from its organic extract, exhibited activity against aquaculture bacteria because of their fatty acids longer than 10 carbon atoms in chain length –which apparently induce lysis of bacterial protoplasts. The ability of fatty acids at large to interfere with bacterial growth and survival has been known for quite some time, but recent structure-function relationship studies suggest that said ability depends on both their chain length and degree of unsaturation. Such compounds as cholesterol can antagonize antimicrobial features [53] so both composition and concentration of free lipids should be taken into account [72]. Among microalgal-derived oxylipins, the antibacterial activities of polyunsaturated aldehydes deserve a special ention. Such compounds are synthesized by diatoms, e.g. *S. costatum* and *Thalassiosira rotula*. One illustrative example is

available target only viral proteins.

*6.3.2. Antibacterial activity* 

**Table 5.** Anticancer activity of the nitrate stressed promising algal species using EACC and HepG2 cell lines [43]

#### *6.3.1. Antiviral activity*

A number of infectious diseases caused by viruses have emerged (and re-emerged) in recent years. Although several antiviral drugs have been specifically developed, drug-resistant mutations are constantly occuring – so new antiviral active principles are necessary, especially those from sources that do not constitute (or are exposed to) viral pools. This is why microalgae have received a strong attention as potential suppliers of antiviral agents [58]; Viral growth is generally divided into three stages, and antiviral action may take place at a single or more stages: Stage I, which consists on adsorption and invasion of cells; Stage II, or eclipse phase, during which the cell is forced to synthesize multiple copies of said virus; and Stage III, or maturity and release of virus particles. For instance, the anti-HSV activity of the antiviral compound acyclovir® is expressed at stage II, but the anti-HSV factor from *Dunaliella* sp. inactivates the viral function at stage I. Sulphated exopolysaccharides from marine microalgae have been claimed to interfere with Stage I of some enveloped viruses they offer competitive advantages because of their broad antiviral spectrum against e.g. HSV and HIV-1 [59]. Apparently, their inhibitory effect arises from interaction with the positive charges on the virus or on the cell surface – which prevents penetration of the former into the host cells; they may also selectively inhibit reverse transcriptase in the case of HIV, thus hampering production of new viral particles after infection yet the exact step during viral replication when they act remains to be elucidated. Antiviral highly sulfated polysaccharides from several species of red microalgae consist mainly of xylose, glucose and galactose [60]; they are unusually stable when exposed to extreme pH and temperature [61]. Despite their successful antiviral performance, the metabolic pathways leading to sulfated polysaccharides are still poorly known. Their secretion by unicellular red algae was originally characterized via radiolabeling – which howed biosynthesis of the carbon chain, and sulfation of the resulting polysaccharide to occur in the Golgi apparatus [62]; these findings were confirmed in *Porphyridium* sp. [63] and other red microalgae [64]. More recently, [65] used 14C pulse-chase experiments and ultrastructural microscopy to conclude that brefeldin A–a membrane-traffic inhibitor of the Golgi apparatus, decreases the contents of the bound and the soluble forms of polysaccharides, while inhibiting cell-wall binding of polysaccharides to a greater extent than its soluble counterpart (in both actively growing and resting cells). Discovery of small molecules that can specifically disrupt a particular protein-protein interface remains a challenge-but is of a particular interest in virology, since the antiviral drugs currently available target only viral proteins.

#### *6.3.2. Antibacterial activity*

290 Plant Science

lines [43]

*6.3.1. Antiviral activity* 

consumers use via self-prescription [56]. However, a key factor for their eventual economic feasibility is the possibility of operating large photobioreactors under aseptic conditions, which

**Table 5.** Anticancer activity of the nitrate stressed promising algal species using EACC and HepG2 cell

A number of infectious diseases caused by viruses have emerged (and re-emerged) in recent years. Although several antiviral drugs have been specifically developed, drug-resistant mutations are constantly occuring – so new antiviral active principles are necessary, especially those from sources that do not constitute (or are exposed to) viral pools. This is why microalgae have received a strong attention as potential suppliers of antiviral agents [58]; Viral growth is generally divided into three stages, and antiviral action may take place at a single or more stages: Stage I, which consists on adsorption and invasion of cells; Stage II, or eclipse phase, during which the cell is forced to synthesize multiple copies of said virus; and Stage III, or maturity and release of virus particles. For instance, the anti-HSV activity of the antiviral compound acyclovir® is expressed at stage II, but the anti-HSV factor from *Dunaliella* sp. inactivates the viral function at stage I. Sulphated exopolysaccharides from marine microalgae have been claimed to interfere with Stage I of some enveloped viruses they offer competitive advantages because of their broad antiviral spectrum against e.g. HSV and HIV-1 [59]. Apparently, their inhibitory effect arises from interaction with the positive charges on the virus or on the cell surface – which prevents penetration of the former into the host cells; they may also selectively inhibit reverse transcriptase in the case of HIV, thus hampering production of new viral particles after infection yet the exact step during viral replication when they act remains to be elucidated. Antiviral highly sulfated polysaccharides from several species of red microalgae consist mainly of xylose, glucose and galactose [60]; they are unusually stable when exposed to extreme pH and temperature [61]. Despite their successful antiviral performance, the metabolic pathways leading to sulfated polysaccharides are still poorly known. Their secretion by unicellular red algae was originally characterized via radiolabeling – which howed biosynthesis of the carbon chain, and sulfation of the resulting polysaccharide to

are able to produce biomass and metabolites to sufficiently high levels [57].

Most efforts were devoted to the study of antibiotic resistance in bacteria for several reasons: (i) bacterial infections are responsible for most community-acquired and nosocomial infections; (ii) the large and expanding number of antibacterial classes offers a more diverse range of resistance mechanisms; and (iii) the ability to move bacterial resistance determinants into standard, well-characterized bacterial strains facilitates more detailed studies of the underlying molecular mechanisms [66]. Pratt et al. [67] isolated the first antibacterial compound from a microalga, *Chlorella*; a mixture of fatty acids, viz. chlorellin, was found to be responsible for that inhibitory activity against both Gram+ and Grambacteria. Research aimed at identifying antibacterial active principles produced by microalgae has meanwhile boomed [68]. This realisation arose e.g. from the risk associated with several multidrug-resistant *Staphylococcus aureus* (MRSA) strains, which have been causing an increased concern in healthcare institutions worldwide – since they are not susceptible to most conventional antibiotics. Hence, discovery of novel antibacterial compounds following distinct biochemical mechanisms of action is urged. Antibiotics are typically less effective against Gram- bacteria because of their complex, multilayered cell wall structure – which makes it more difficult for the active compound to penetrate them [69]; this justifies why the antibacterial activity of the supernatant (and methanolic extracts) is more potent against Gram+ than Gram- bacteria [68,70]. The exact mechanism of action of fatty acids remains unknown: they may act upon multiple cellular targets, even though cell membranes are the most probable ones – as membrane damage will likely lead to cell leakage and reduction of nutrient uptake, besides inhibiting cellular respiration; conversely, Desbois [71] claimed a peroxidative process. Furthermore, compounds synthesized by *Scenedesmus costatum*, and partially purified from its organic extract, exhibited activity against aquaculture bacteria because of their fatty acids longer than 10 carbon atoms in chain length –which apparently induce lysis of bacterial protoplasts. The ability of fatty acids at large to interfere with bacterial growth and survival has been known for quite some time, but recent structure-function relationship studies suggest that said ability depends on both their chain length and degree of unsaturation. Such compounds as cholesterol can antagonize antimicrobial features [53] so both composition and concentration of free lipids should be taken into account [72]. Among microalgal-derived oxylipins, the antibacterial activities of polyunsaturated aldehydes deserve a special ention. Such compounds are synthesized by diatoms, e.g. *S. costatum* and *Thalassiosira rotula*. One illustrative example is

decadienal–probably derived from (the polyunsaturated) arachidonic acid (C20:4 n-3), which exhibits a strong activity against such important human pathogens as MRSA and *Haemophilus influenza* – with MIC values of 7.8 and 1.9 µg/mL, respectively, and well as against *E. coli* and *Pseudomonas aeruginosa*, and *S. aureus and Staphylococcus epidermidis*  (Gram- and Gram+ bacteria, respectively). Furthermore, it impairs growth of diverse marine bacteria, such as (the Gram-) *Aeromonas hydrophila*, *L. anguillarum, Alteromonas haloplankti, Photobacterium phosphoreum* and *Psychrobacter immobilis*, and the (Gram+) *Planococcus citreus*  and *Micrococcus luteus* [73].

Microalgal Biotechnology: Prospects and Applications 293

**Figure 8.** The efficacy of (A) *Nostoc muscorum,* (B) *Oscillatoria* sp and (C) *Spirulina platensis* culture filtrates (40 ml.L-1) in suppressing the fungal mycelia growth diameter of *Cercospora beticola* [77]

Culture filtrates of Microcoleus vaginatus inhibited hatching of *Meloidogyne incognita* eggs and killed second stage juveniles. Microalgal metabolites have attracted attention, because they are a resource for toxins, and potential new drugs [80]. Shawky et al. [81] studied the nematicidal effect of nine culture filtrates of algal strains (*Nostoc muscorum*, *Anabaena flos* 

#### *6.3.3. Antifungal activity*

Algae are one of the chief biological agents that have been studied for the control of fungi plant pathogens [74]. Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with diverse biological activities such as antibacterial, antifungal and antiviral activity [75]. These biologically active compounds include antibiotics and toxins [76]. Hussien *et al.* [77] screened the effect of culture filtrates of nine algal strains (*Anabaena flosaquae*, *Anabaena oryzae*, *Chlorella vulgaris, Nostoc muscorum*, *Nostoc humifusu, Oscillatoria* sp.*, Phormedium fragile*, *Spirulina platensis* and *Wollea saccata*) at concentrations of 10, 20, 30 and 40% on mycelium growth of the plant pathogenic fungi *Cercospora beticola* causing leaf spot disease in sugar beet comparing with different concentrations of the synthetic fungicide Topsin M70 (100, 200, 300 and 400 ppm). They found that generally, all the algal culture filtrates reduced the fungal mycelium growth but the highest fungal mycelium growth inhibition percentages were achieved by *Spirulina platensis*, *Oscillatoria* sp. and *Nostoc muscorum* (Figure 8) at the concentrations of 30% (100, 100 and 82%, respectively) and at 40% (100, 100 and 100%, respectively). While, the chemical synthetic fungicide Topsin M70 completely inhibited the fungal mycelium growth at the concentrations of 200, 300 and 400 ppm. Fungal spores production (number of spores) was completely inhibited by the previous three algal culture filtrates at 300 and 400 ppm particularly at the concentration of 40%. Same results were obtained by Topsin M70. The antifungal activity of the algal culture filtrates has been attributed to the presence of bioactive compounds i.e. total phenolic compounds, total saponins and alkaloids in the algal culture filtrates.

#### **6.4. Nematicidal activity**

Root-knot nematodes, *Meloidogyne* spp., are among the most damaging nematodes in agriculture, causing an estimated US\$ 100 billion loss/year worldwide [78]. The application of chemical nematicides has been found as an effective measure for controlling nematodes but they have toxic residual effect on the environment particularly on non-target organisms and human health. In addition, the use of chemical nematicides is prohibited in organic farming. Nematicidal potential of cyanobacteria has remained unexplored except for a few reports, which suggest that endospores of Microcoleus and *Oscillatoria* spp. killed nematodes [79].

Microalgal Biotechnology: Prospects and Applications 293

292 Plant Science

and *Micrococcus luteus* [73].

*6.3.3. Antifungal activity* 

culture filtrates.

nematodes [79].

**6.4. Nematicidal activity** 

decadienal–probably derived from (the polyunsaturated) arachidonic acid (C20:4 n-3), which exhibits a strong activity against such important human pathogens as MRSA and *Haemophilus influenza* – with MIC values of 7.8 and 1.9 µg/mL, respectively, and well as against *E. coli* and *Pseudomonas aeruginosa*, and *S. aureus and Staphylococcus epidermidis*  (Gram- and Gram+ bacteria, respectively). Furthermore, it impairs growth of diverse marine bacteria, such as (the Gram-) *Aeromonas hydrophila*, *L. anguillarum, Alteromonas haloplankti, Photobacterium phosphoreum* and *Psychrobacter immobilis*, and the (Gram+) *Planococcus citreus* 

Algae are one of the chief biological agents that have been studied for the control of fungi plant pathogens [74]. Various strains of cyanobacteria are known to produce intracellular and extracellular metabolites with diverse biological activities such as antibacterial, antifungal and antiviral activity [75]. These biologically active compounds include antibiotics and toxins [76]. Hussien *et al.* [77] screened the effect of culture filtrates of nine algal strains (*Anabaena flosaquae*, *Anabaena oryzae*, *Chlorella vulgaris, Nostoc muscorum*, *Nostoc humifusu, Oscillatoria* sp.*, Phormedium fragile*, *Spirulina platensis* and *Wollea saccata*) at concentrations of 10, 20, 30 and 40% on mycelium growth of the plant pathogenic fungi *Cercospora beticola* causing leaf spot disease in sugar beet comparing with different concentrations of the synthetic fungicide Topsin M70 (100, 200, 300 and 400 ppm). They found that generally, all the algal culture filtrates reduced the fungal mycelium growth but the highest fungal mycelium growth inhibition percentages were achieved by *Spirulina platensis*, *Oscillatoria* sp. and *Nostoc muscorum* (Figure 8) at the concentrations of 30% (100, 100 and 82%, respectively) and at 40% (100, 100 and 100%, respectively). While, the chemical synthetic fungicide Topsin M70 completely inhibited the fungal mycelium growth at the concentrations of 200, 300 and 400 ppm. Fungal spores production (number of spores) was completely inhibited by the previous three algal culture filtrates at 300 and 400 ppm particularly at the concentration of 40%. Same results were obtained by Topsin M70. The antifungal activity of the algal culture filtrates has been attributed to the presence of bioactive compounds i.e. total phenolic compounds, total saponins and alkaloids in the algal

Root-knot nematodes, *Meloidogyne* spp., are among the most damaging nematodes in agriculture, causing an estimated US\$ 100 billion loss/year worldwide [78]. The application of chemical nematicides has been found as an effective measure for controlling nematodes but they have toxic residual effect on the environment particularly on non-target organisms and human health. In addition, the use of chemical nematicides is prohibited in organic farming. Nematicidal potential of cyanobacteria has remained unexplored except for a few reports, which suggest that endospores of Microcoleus and *Oscillatoria* spp. killed

**Figure 8.** The efficacy of (A) *Nostoc muscorum,* (B) *Oscillatoria* sp and (C) *Spirulina platensis* culture filtrates (40 ml.L-1) in suppressing the fungal mycelia growth diameter of *Cercospora beticola* [77]

Culture filtrates of Microcoleus vaginatus inhibited hatching of *Meloidogyne incognita* eggs and killed second stage juveniles. Microalgal metabolites have attracted attention, because they are a resource for toxins, and potential new drugs [80]. Shawky et al. [81] studied the nematicidal effect of nine culture filtrates of algal strains (*Nostoc muscorum*, *Anabaena flos*  *aquae*, *Anabaena oryzae*, *Chlorella vulgaris, Wollea saccata, Phormedium fragile*, *Oscillatoria* sp.*, Nostoc humifusum* and *Spirulina platensis*), *Azolla pinnata* aqueous extract filtrate (1:2w/v) and compost watery extract filtrate (1:5w/v) in controlling the population of the root knot nematode, *Meloidogyne incognita* in cucumber under both laboratory and greenhouse conditions. Laboratory experiment revealed that high juvenile mortality percentage occurred during all the exposure periods of all treatments, the best results were after 72 hr exposure. Only five cyanobacterial strains, namely, *Spirulina platensis*, *Oscillatoria* sp., *Anabaena oryzae*, *Nostoc muscorum* and *Phormedium fragile*, followed by compost watery extract, significantly increased juveniles mortality over 70% at the highest concentration of 1:10 (84.3, 80.4, 78.9, 75.4, 72.5 and 70.1%, respectively). A*zolla pinnata* aqueous extract filtrate achieved 69.8% at the same concentration while, *Anabaena flos aquae* and *Chlorella vulgaris* recorded the lowest effect on mortality percentage (52.1 and 40.1%, respectively) at the concentration of 1:10. In the greenhouse experiment, the combination of mixing five algal culture filtrates of *S. platensis*, *Oscillatoria* sp., *A. oryzae*, *N. muscorum* and *P. fragile* with *A. pinnata* aqueous extract filtrate and compost extract achieved the highest reduction in the number of the 2*nd* stage juveniles in soil, the numbers of galls, developmental stages, females, egg masses, egg numbers/egg mass in roots of cucumber plants comparing with the individual treatment and the non treated control. In addition, all combinations significantly improved fresh weight of roots and shoots and increased the yield of cucumber plants.

Microalgal Biotechnology: Prospects and Applications 295

available for the parasite transmission. Therefore it may be a potential source of effective

**Figure 9.** Section in the hermaphrodite gland of *Biomphalaria alexandrina* snail: control (A), exposed to 0.07% (B) and to 0.17% (C) concentrations of *Spirulina platensis* culture filtrate. G=Spermatogonia,

Microalgae have found commercial applications as natural sources of valuable macromolecules, including carotenoids, long-chain polyunsaturated fatty acids, and phycocolloids. As photoautotrophs, their simple growth requirements make them attractive for bioprocesses aimed at producing high added-value compounds that are in large demand by the pharmaceutical market. The productivity and biochemical composition of microalgae depend strongly on the mode of cultivation, medium composition, and nutrient profile. Consequently, numerous efforts aimed at elucidating the practical impacts of the aforementioned parameters have been developed [56]. Thus, there is a growing interest in

the area of research on the positive effect of algae on human health and other benefits.

The first use of microalgae by humans dates back 2000 years to the Chinese, who used *Nostoc* to survive during famine. Microalgae for human nutrition are nowadays marketed in different forms such as tablets, capsules and liquids. They can also be incorporated into pastas, snack foods, candy bars or gums, and beverages [85-86]. Owing to their diverse chemical properties, they can act as a nutritional supplement or represent a source of natural food colorants. The commercial applications are dominated by four strains: *Arthrospira, Chlorella, Dunaliella salina* and *Aphanizomenon flos-aquae*. *Arthrospira* is used in human nutrition because of its high protein content and its excellent nutritive value [87-88]). In

T=Spermatid, Z=Spermatozoa, I= Oogonia, O=Oocyte V= vacules D=degeneration [84]

**7. Other applications and products from microalgae** 

**7.1. Food** 

compounds for control of *Schistosoma mansoni*.

#### **6.5. Mollscicidal activity**

The snail intermediate hosts of schistosomiasis are the sites of intense multiplication of this parasite, thus their control strategies are considered a priority of the reduction of schistosomiasis transmission [82]. Although chemical molluscicides are to certain extent quite successful in curbing the disease concerned. However, in view of their side effects, interest in environmentally friendly approaches and use of biological control agents have been revived [83]. Mostafa and Gawish [84] stated that the algal culture filtrate of *Spirulina platensis* was proved for its mollscicidal activity against *Biomphalaria alexandrina* snails the intermediate host of *Schistosoma mansoni* in Egypt that accommodates their survival and fecundity and also for its effect on hatchability of snail's eggs and viability of the free living larval stages of the parasite (miracidia and cercariae). The culture filtrate proved to have a lethal effect on snails with 90 LC 0.23% of the filtrate. *B. alexandrina* snails stopped egg laying after one week of continuous exposure to the sublethal concentrations 0.19 and 0.17%, while those exposed to 0.07% laid few ones. It could be due to the phytochemical constituents of the culture filtrate i.e. total phenolic compounds, alkaloids and total saponins. This was confirmed by histological examination that showed a severe damage in the hermaphrodite gland cells of snails (Figure 9) exposed to these concentrations. *B. alexandrina* eggs of 3 and 7 days old failed to hatch post exposure to 0.1% of algal filtrate, while 22% and 10% only hatched after exposure to 0.05% of this filtrate. Free cell culture filtrate shows also marked miracidicidal and cercaricidal activities as 2% of this filtrate killed most of these organisms within 15 minutes of exposure. It is concluded that the byproducts of the blue-green alga *Spirulina platensis* has a lethal effect against adult *B. alexandria* snails, reduced or stopped their oviposition, hence minimize the snail populations available for the parasite transmission. Therefore it may be a potential source of effective compounds for control of *Schistosoma mansoni*.

**Figure 9.** Section in the hermaphrodite gland of *Biomphalaria alexandrina* snail: control (A), exposed to 0.07% (B) and to 0.17% (C) concentrations of *Spirulina platensis* culture filtrate. G=Spermatogonia, T=Spermatid, Z=Spermatozoa, I= Oogonia, O=Oocyte V= vacules D=degeneration [84]

## **7. Other applications and products from microalgae**

Microalgae have found commercial applications as natural sources of valuable macromolecules, including carotenoids, long-chain polyunsaturated fatty acids, and phycocolloids. As photoautotrophs, their simple growth requirements make them attractive for bioprocesses aimed at producing high added-value compounds that are in large demand by the pharmaceutical market. The productivity and biochemical composition of microalgae depend strongly on the mode of cultivation, medium composition, and nutrient profile. Consequently, numerous efforts aimed at elucidating the practical impacts of the aforementioned parameters have been developed [56]. Thus, there is a growing interest in the area of research on the positive effect of algae on human health and other benefits.

#### **7.1. Food**

294 Plant Science

**6.5. Mollscicidal activity** 

*aquae*, *Anabaena oryzae*, *Chlorella vulgaris, Wollea saccata, Phormedium fragile*, *Oscillatoria* sp.*, Nostoc humifusum* and *Spirulina platensis*), *Azolla pinnata* aqueous extract filtrate (1:2w/v) and compost watery extract filtrate (1:5w/v) in controlling the population of the root knot nematode, *Meloidogyne incognita* in cucumber under both laboratory and greenhouse conditions. Laboratory experiment revealed that high juvenile mortality percentage occurred during all the exposure periods of all treatments, the best results were after 72 hr exposure. Only five cyanobacterial strains, namely, *Spirulina platensis*, *Oscillatoria* sp., *Anabaena oryzae*, *Nostoc muscorum* and *Phormedium fragile*, followed by compost watery extract, significantly increased juveniles mortality over 70% at the highest concentration of 1:10 (84.3, 80.4, 78.9, 75.4, 72.5 and 70.1%, respectively). A*zolla pinnata* aqueous extract filtrate achieved 69.8% at the same concentration while, *Anabaena flos aquae* and *Chlorella vulgaris* recorded the lowest effect on mortality percentage (52.1 and 40.1%, respectively) at the concentration of 1:10. In the greenhouse experiment, the combination of mixing five algal culture filtrates of *S. platensis*, *Oscillatoria* sp., *A. oryzae*, *N. muscorum* and *P. fragile* with *A. pinnata* aqueous extract filtrate and compost extract achieved the highest reduction in the number of the 2*nd* stage juveniles in soil, the numbers of galls, developmental stages, females, egg masses, egg numbers/egg mass in roots of cucumber plants comparing with the individual treatment and the non treated control. In addition, all combinations significantly improved fresh weight of roots and shoots and increased the yield of cucumber plants.

The snail intermediate hosts of schistosomiasis are the sites of intense multiplication of this parasite, thus their control strategies are considered a priority of the reduction of schistosomiasis transmission [82]. Although chemical molluscicides are to certain extent quite successful in curbing the disease concerned. However, in view of their side effects, interest in environmentally friendly approaches and use of biological control agents have been revived [83]. Mostafa and Gawish [84] stated that the algal culture filtrate of *Spirulina platensis* was proved for its mollscicidal activity against *Biomphalaria alexandrina* snails the intermediate host of *Schistosoma mansoni* in Egypt that accommodates their survival and fecundity and also for its effect on hatchability of snail's eggs and viability of the free living larval stages of the parasite (miracidia and cercariae). The culture filtrate proved to have a lethal effect on snails with 90 LC 0.23% of the filtrate. *B. alexandrina* snails stopped egg laying after one week of continuous exposure to the sublethal concentrations 0.19 and 0.17%, while those exposed to 0.07% laid few ones. It could be due to the phytochemical constituents of the culture filtrate i.e. total phenolic compounds, alkaloids and total saponins. This was confirmed by histological examination that showed a severe damage in the hermaphrodite gland cells of snails (Figure 9) exposed to these concentrations. *B. alexandrina* eggs of 3 and 7 days old failed to hatch post exposure to 0.1% of algal filtrate, while 22% and 10% only hatched after exposure to 0.05% of this filtrate. Free cell culture filtrate shows also marked miracidicidal and cercaricidal activities as 2% of this filtrate killed most of these organisms within 15 minutes of exposure. It is concluded that the byproducts of the blue-green alga *Spirulina platensis* has a lethal effect against adult *B. alexandria* snails, reduced or stopped their oviposition, hence minimize the snail populations

The first use of microalgae by humans dates back 2000 years to the Chinese, who used *Nostoc* to survive during famine. Microalgae for human nutrition are nowadays marketed in different forms such as tablets, capsules and liquids. They can also be incorporated into pastas, snack foods, candy bars or gums, and beverages [85-86]. Owing to their diverse chemical properties, they can act as a nutritional supplement or represent a source of natural food colorants. The commercial applications are dominated by four strains: *Arthrospira, Chlorella, Dunaliella salina* and *Aphanizomenon flos-aquae*. *Arthrospira* is used in human nutrition because of its high protein content and its excellent nutritive value [87-88]). In addition, this microalga has various possible healthpromoting effects: the alleviation of hyperlipidemia, suppression of hypertension, protection against renal failure, growth promotion of intestinal *Lactobacillus*, and suppression of elevated serum glucose level [85- 86]. A significant amount of *Arthrospira* production is realized in China and India. *Chlorella*  can also be used as a food additive owing to the taste- and flavour-adjusting actions of its coloring agent [85]. *D. salina* is exploited for its β-carotene content that can reach 14% of dry weight [89]. For human consumption, Cognis Nutrition and Health, the world's largest producer of this strain, offers *Dunaliella* powder as an ingredient of dietary supplements and functional foods. The last major commercial strain application is *A. flos-aquae*. According to many research studies, used alone or in combination with other nutraceuticals and natural food products, *A. flos-aquae* promotes good overall health [85,90].The consumption of *Arthrospira (Spirulina)* by the Kanembu was also reported by Delisle et al. [91] in a survey of household food consumption and nutritional adequacy in Wadi zones of Chad. Table (6) presents a comparison of the general compositions of human food sources with that of different microalgae.

Microalgal Biotechnology: Prospects and Applications 297

*Skeletonema* and *Thalassiosira* [85,94]. Many nutritional and toxicological evaluations have proved the suitability of algal biomass as feed supplement [92]. *Arthrospira* is largely used in this domain and concerns many types of animal: cats, dogs, aquarium fish, ornamental birds, horses, cows and breeding bulls. Algae positively affect the physiology (by providing a large profile of natural vitamins, minerals, and essential fatty acids; improved immune response and fertility; and better weight control) and their external appearance (resulting in healthy skin and a lustrous coat) of animals. In poultry rations, algae up to a level of 5-10% can be used safely as partial replacement for conventional proteins. Prolonged feeding of algae at higher concentrations produces adverse effects. The yellow color of broiler skin and shanks as well as of egg yolk is the most important characteristic that can be influenced by

Humans have practiced agriculture for more than 10,000 years, but only in the past 50 years or so have farmers become heavily dependent on synthetic chemical fertilizers and pesticides. It contributes to numerous forms of environmental degradation, including air and water pollution, soil depletion and diminishing biodiversity. Synthetic chemical pesticides and fertilizers are polluting soil, water, and air, harming both the environment and human health. Soil is eroding much faster than it can be replenished—taking with it the land's fertility and nutrients that nourish both plants and those who eat them. Chemical fertilizers can gradually increase the acidity of the soil until it begins to impede plant growth. Chemically fertilized plots also show less biologic activity in the soil food web (the microscopic organisms that make up the soil ecosystem) than do plots fertilized organically with manure or other biologic sources of fertility [95]. The best way, however, is to use as much as possible microbial products, functional bio-fertilizers and bio-controllers and reduce the amount of the use of chemical fertilizers or pesticides. Heterocystous cyanobacteria and several nonheterocystous cyanobacteria are known for their ability to fix atmospheric nitrogen. The fertility of many tropical rice field soils has been mainly attributed to the activity of nitrogen-fixing cyanobacteria. An estimation showed that more than 18 kg N ha-1 year-1 was added to the soils by cyanobacteria. Inoculation of cyanobacteria to increase the fertility of soils has been successfully attempted. Recently, nitrogen-fixing cyanobacteria have been reported to dominate desert crusts worldwide. This is believed to contribute significantly to the fertility of desert soils and may eventually facilitate vegetation of deserts [96]. Algae as biofertilizers are a promising alternative to avoid soil pollution caused by agrochemicals. Also, they recover the nutrients content to soil as they secrete exo-polysaccharides that improve soil structure and bio-active substances that enhance the plant growth. Algae are known to be one of the most promising sources as bio-control agents of any residues, thereby having positive impact on human health [97]. Microorganisms play an important role in various chemical transformations of soils and thus, influence the availability of major nutrients like nitrogen, phosphorus, potassium and sulphur to the plants. Cyanobacteria and phosphate-solubilizing bacteria were used as biofertilizers to increase crop production [98].The cyanobacterial ability to mobilize insoluble forms of inorganic phosphates is evident from the finding of kleiner and Harper

feeding algae [92].

**7.3. Agricultural purposes** 


**Table 6.** General composition (%) of dry matter of different human food sources and algae [92].

#### **7.2. Feed**

Microalgae can be incorporated into the feed for a wide variety of animals ranging from fish (aquaculture) to pets and farm animals. In fact, 30% of the current world algal production is sold for animal feed applications [92] and over 50% of the current world production of *Arthrospira* is used as feed supplement [85]. In 1999, the production of microalgae for aquaculture reached 1000 t (62% for molluscs, 21% for shrimps, and 16% for fish) for a global world aquaculture production of 43x106 t of plants and animals [93]. The importance of algae in this domain is not surprising as they are the natural food source of these animals. The main applications of microalgae for aquaculture are associated with nutrition, being used fresh (as sole component or as food additive to basic nutrients) for coloring the flesh of salmonids and for inducing other biological activities. The most frequently used species are *Chlorella, Tetraselmis*, *Isochrysis*, *Pavlova*, *Phaeodactylum*, *Chaetoceros*, *Nannochloropsis,*  *Skeletonema* and *Thalassiosira* [85,94]. Many nutritional and toxicological evaluations have proved the suitability of algal biomass as feed supplement [92]. *Arthrospira* is largely used in this domain and concerns many types of animal: cats, dogs, aquarium fish, ornamental birds, horses, cows and breeding bulls. Algae positively affect the physiology (by providing a large profile of natural vitamins, minerals, and essential fatty acids; improved immune response and fertility; and better weight control) and their external appearance (resulting in healthy skin and a lustrous coat) of animals. In poultry rations, algae up to a level of 5-10% can be used safely as partial replacement for conventional proteins. Prolonged feeding of algae at higher concentrations produces adverse effects. The yellow color of broiler skin and shanks as well as of egg yolk is the most important characteristic that can be influenced by feeding algae [92].

#### **7.3. Agricultural purposes**

296 Plant Science

different microalgae.

**7.2. Feed** 

addition, this microalga has various possible healthpromoting effects: the alleviation of hyperlipidemia, suppression of hypertension, protection against renal failure, growth promotion of intestinal *Lactobacillus*, and suppression of elevated serum glucose level [85- 86]. A significant amount of *Arthrospira* production is realized in China and India. *Chlorella*  can also be used as a food additive owing to the taste- and flavour-adjusting actions of its coloring agent [85]. *D. salina* is exploited for its β-carotene content that can reach 14% of dry weight [89]. For human consumption, Cognis Nutrition and Health, the world's largest producer of this strain, offers *Dunaliella* powder as an ingredient of dietary supplements and functional foods. The last major commercial strain application is *A. flos-aquae*. According to many research studies, used alone or in combination with other nutraceuticals and natural food products, *A. flos-aquae* promotes good overall health [85,90].The consumption of *Arthrospira (Spirulina)* by the Kanembu was also reported by Delisle et al. [91] in a survey of household food consumption and nutritional adequacy in Wadi zones of Chad. Table (6) presents a comparison of the general compositions of human food sources with that of

Commodity Protein Carbohydrate Lipid Bakers' yeast 39 38 1 Meat 43 1 34 Milk 26 38 28 Rice 8 77 2 Soybean 37 30 20 *Anabaena cylindrica* 43-56 25-30 4-7 *Chlamydomonas rheinhardii* 48 17 21 *Chlorella vulgaris* 51-58 12-17 14-22 *Dunaliella salina* 57 32 6 *Porphyridium cruentum* 28-39 40-57 9-14 *Scenedesmus obliquus* 50-56 10-17 12-14 *Spirulina maxima* 60-71 13-16 6-7 *Synechococcus sp.* 63 15 11 **Table 6.** General composition (%) of dry matter of different human food sources and algae [92].

Microalgae can be incorporated into the feed for a wide variety of animals ranging from fish (aquaculture) to pets and farm animals. In fact, 30% of the current world algal production is sold for animal feed applications [92] and over 50% of the current world production of *Arthrospira* is used as feed supplement [85]. In 1999, the production of microalgae for aquaculture reached 1000 t (62% for molluscs, 21% for shrimps, and 16% for fish) for a global world aquaculture production of 43x106 t of plants and animals [93]. The importance of algae in this domain is not surprising as they are the natural food source of these animals. The main applications of microalgae for aquaculture are associated with nutrition, being used fresh (as sole component or as food additive to basic nutrients) for coloring the flesh of salmonids and for inducing other biological activities. The most frequently used species are *Chlorella, Tetraselmis*, *Isochrysis*, *Pavlova*, *Phaeodactylum*, *Chaetoceros*, *Nannochloropsis,*  Humans have practiced agriculture for more than 10,000 years, but only in the past 50 years or so have farmers become heavily dependent on synthetic chemical fertilizers and pesticides. It contributes to numerous forms of environmental degradation, including air and water pollution, soil depletion and diminishing biodiversity. Synthetic chemical pesticides and fertilizers are polluting soil, water, and air, harming both the environment and human health. Soil is eroding much faster than it can be replenished—taking with it the land's fertility and nutrients that nourish both plants and those who eat them. Chemical fertilizers can gradually increase the acidity of the soil until it begins to impede plant growth. Chemically fertilized plots also show less biologic activity in the soil food web (the microscopic organisms that make up the soil ecosystem) than do plots fertilized organically with manure or other biologic sources of fertility [95]. The best way, however, is to use as much as possible microbial products, functional bio-fertilizers and bio-controllers and reduce the amount of the use of chemical fertilizers or pesticides. Heterocystous cyanobacteria and several nonheterocystous cyanobacteria are known for their ability to fix atmospheric nitrogen. The fertility of many tropical rice field soils has been mainly attributed to the activity of nitrogen-fixing cyanobacteria. An estimation showed that more than 18 kg N ha-1 year-1 was added to the soils by cyanobacteria. Inoculation of cyanobacteria to increase the fertility of soils has been successfully attempted. Recently, nitrogen-fixing cyanobacteria have been reported to dominate desert crusts worldwide. This is believed to contribute significantly to the fertility of desert soils and may eventually facilitate vegetation of deserts [96]. Algae as biofertilizers are a promising alternative to avoid soil pollution caused by agrochemicals. Also, they recover the nutrients content to soil as they secrete exo-polysaccharides that improve soil structure and bio-active substances that enhance the plant growth. Algae are known to be one of the most promising sources as bio-control agents of any residues, thereby having positive impact on human health [97]. Microorganisms play an important role in various chemical transformations of soils and thus, influence the availability of major nutrients like nitrogen, phosphorus, potassium and sulphur to the plants. Cyanobacteria and phosphate-solubilizing bacteria were used as biofertilizers to increase crop production [98].The cyanobacterial ability to mobilize insoluble forms of inorganic phosphates is evident from the finding of kleiner and Harper [99] who reported more extractable phosphates in soils with cyanobacterial cover than in nearby soils without cover. Cyanobacteria can fix about 25 kg N/ha/season. Apart from nitrogen fixation, inoculation with cyanobacteria is also reported to reduce considerably the total sulphides and ferrous iron content of the soil. Blue-green algae constitute an important group of microorganism capable of nitrogen fixation. Most of the species possess nitrogen fixation ability to the order Nostocales and Stigonematales. Over 100 species of blue-green algae are known to fix atmospheric nitrogen. These have been found to be very effective on the rice and banana plantation. In field condition, overall increase in the gram yield of rice is amounted to about 586 kg/ha. In case of crops other than rice, algalization increased nearly 34 per cent yield. India is one of the countries where agro-chemical conditions appear to be favourable where blue-green algae technology has been put forward. In some parts of the country, production of BGA inoculants has been commercialised. Producing inoculum in artificially controlled conditions is well defined, but relatively expensive. On the other hand open-air soil culture is simpler, less expensive and easily adaptable by the farmers. Field scale production of algae biofertilizer is also possible. 20-25 kg dry algae can be obtained on 40 m field. Adopting this method, 15 t/ha of wet BGA can be obtained by the farmers. Farmers can also produce algae for countryard of the house [96]. Blue-green algal extracts comprise a great number of bioactive compounds that influence plant growth and development. They mostly contain growth phyto-regulators like gibberellins, auxin, cytokinin, ethylene and abscisic acid [100]. This group of microorganisms have been reported to benefit plants by producing growth promoting regulators resemble gibberellin and auxin, vitamins, amino acids, polypeptides, antibacterial and antifungal substances that exert phytopathogen biocontrol and polymers especially exopolysaccharides that were reported to enhance growth and productivity of plants like *Daucus carota* [101], *Santalum album* [102], *Oryzae sativa* [103], *Lilium alexandrae* hort [104] and *Beta vulgaris* L.[100]. Nonnitrogen fixing cyanobacteria can enrich phosphorus and potassium contents in soils, laying indirect major role in plant growth promotion [105]. Cyanobacteria also enhance the soil biological activity in terms of increasing the total bacterial, total cyanobacterial counts, CO2 evolution, dehydrogenase and nitrogenase activities. Many researches suggested that up to 50% of the recommended dose of the mineral nitrogen fertilizers could be saved by using some species of nitrogen fixing cyanobacteria. The obtained results emphasized the prospects and potentials of using cyanobacteria biofertilizers as renewable natural nitrogen resources for many crops. They are none polluting, inexpensive, utilize renewable resources (inorganic nutrients and atmospheric CO2) in addition to their ability in using free available solar energy, atmospheric nitrogen and water [106-108].

Microalgal Biotechnology: Prospects and Applications 299

production of high value products such as pigments and high-grade lipids, which are extractable from several species of algae. Brennan and Owende [110] also mention high value products such as animal feed supplements being extractable from the microalgae species *Chlorella*, *Scenedesmus* and *Spirulina*. The urgent need for substantive net reductions in CO2 emissions to the atmosphere can be addressed via biological CO2 mitigation, coupled with transition to more extensive uses of biofuel, nuclear and renewable energy sources. Microalgae have attracted a great deal of attention for CO2 fixation and biofuel production because they can convert CO2 (and supplementary nutrients) into biomass via photosynthesis at much higher rates than conventional biofuel crops can. This biomass may then be transformed into methane or hydrogen, using processes mediated by anaerobic bacteria; an integrated process for hydrothermal production of methane via microalgae has been discussed recently [111-112]. Of particular interest is the production of oils by microalgae because of the ease of their synthesis (a lack of a nitrogen source usually suffices to trigger this form of secondary metabolism). Lipid extraction and re-esterification are accomplished with short-chain alcohols and other by-products of secondary metabolism (i.e. polyunsaturated fatty acids, bcarotenes or polymers [111]. Upon extraction, such oils can be hydrolyzed and then re-esterified with methyl- or ethyl alcohol moieties to obtain biodiesel. Microalga-mediated CO2 fixation and biofuel production can be rendered more sustainable by coupling microalgal biomass production with existing power generation and wastewater treatment infrastructures (Figure 10). Microalgae can utilize low-quality water, such as agricultural runoff or municipal, industrial or agricultural wastewaters, as a source of water for the growth medium as well as a source of nitrogen, phosphorus and minor nutrients

Wastewater nitrogen and phosphorous as microalgae nutrients aquaculture systems involving microalgae production and wastewater treatment (e.g. of amino acids, enzyme, or food industries wastewaters) seems to be quite promising for microalgae growth combined with biological cleaning. This allows nutrition of microalgae by using organic compounds (nitrogen and phosphorous) available in some manufactures wastewater, not containing heavy metals and radioisotopes. Additionally, microalgae can mitigate the effects of sewage effluent and industrial sources of nitrogenous waste such as those originating from water treatment or fish aquaculture and at the same time contributing to biodiversity. Moreover, removing nitrogen and carbon from water, microalgae can help reduce the eutrophication in the aquatic environment. Aslan and Kapdan [114] used *C. vulgaris* for nitrogen and phosphorus removal from wastewater with an average removal efficiency of 72% for nitrogen and 28% for phosphorus (from 3 to 8 mg/L NH4+ and 1.5–3.5 mg/L PO4 \_3). Other widely used microalgae cultures for nutrient removal are *Chlorella* [115] and *Spirulina* species [116]. Nutrient removal capacities of *Nannochloris* [117]), *Botryococcus brauinii* [118] and cyanobacterium *Phormidium bohneri* have also been investigated [119-120]. Environmental applications production of biodiesel and other bio-products from microalgae can be more environmentally sustainable, cost-effective and profitable, if combined with processes such as wastewater and flue gas treatments. In fact various studies demonstrated

[113].

**7.5. Wastewater treatments** 

#### **7.4. Mitigation of CO2: Why algae for CO2 sequestration?**

Many options that have been proposed and that are in use for capturing CO2 can be seen as economically, socially and environmentally short-sighted. A common approach is taking measures to offset any immediate effects, often by simple relocation of the emissions. Injection of flue gases into oceanic or geological sinks is examples of such "end-of-pipe" solutions [109]. Algae cultivation can yield a broad range of useful end products, apart from biofuels. The sequestration of CO2 into algal biomass can become profitable also through the production of high value products such as pigments and high-grade lipids, which are extractable from several species of algae. Brennan and Owende [110] also mention high value products such as animal feed supplements being extractable from the microalgae species *Chlorella*, *Scenedesmus* and *Spirulina*. The urgent need for substantive net reductions in CO2 emissions to the atmosphere can be addressed via biological CO2 mitigation, coupled with transition to more extensive uses of biofuel, nuclear and renewable energy sources. Microalgae have attracted a great deal of attention for CO2 fixation and biofuel production because they can convert CO2 (and supplementary nutrients) into biomass via photosynthesis at much higher rates than conventional biofuel crops can. This biomass may then be transformed into methane or hydrogen, using processes mediated by anaerobic bacteria; an integrated process for hydrothermal production of methane via microalgae has been discussed recently [111-112]. Of particular interest is the production of oils by microalgae because of the ease of their synthesis (a lack of a nitrogen source usually suffices to trigger this form of secondary metabolism). Lipid extraction and re-esterification are accomplished with short-chain alcohols and other by-products of secondary metabolism (i.e. polyunsaturated fatty acids, bcarotenes or polymers [111]. Upon extraction, such oils can be hydrolyzed and then re-esterified with methyl- or ethyl alcohol moieties to obtain biodiesel. Microalga-mediated CO2 fixation and biofuel production can be rendered more sustainable by coupling microalgal biomass production with existing power generation and wastewater treatment infrastructures (Figure 10). Microalgae can utilize low-quality water, such as agricultural runoff or municipal, industrial or agricultural wastewaters, as a source of water for the growth medium as well as a source of nitrogen, phosphorus and minor nutrients [113].

#### **7.5. Wastewater treatments**

298 Plant Science

[99] who reported more extractable phosphates in soils with cyanobacterial cover than in nearby soils without cover. Cyanobacteria can fix about 25 kg N/ha/season. Apart from nitrogen fixation, inoculation with cyanobacteria is also reported to reduce considerably the total sulphides and ferrous iron content of the soil. Blue-green algae constitute an important group of microorganism capable of nitrogen fixation. Most of the species possess nitrogen fixation ability to the order Nostocales and Stigonematales. Over 100 species of blue-green algae are known to fix atmospheric nitrogen. These have been found to be very effective on the rice and banana plantation. In field condition, overall increase in the gram yield of rice is amounted to about 586 kg/ha. In case of crops other than rice, algalization increased nearly 34 per cent yield. India is one of the countries where agro-chemical conditions appear to be favourable where blue-green algae technology has been put forward. In some parts of the country, production of BGA inoculants has been commercialised. Producing inoculum in artificially controlled conditions is well defined, but relatively expensive. On the other hand open-air soil culture is simpler, less expensive and easily adaptable by the farmers. Field scale production of algae biofertilizer is also possible. 20-25 kg dry algae can be obtained on 40 m field. Adopting this method, 15 t/ha of wet BGA can be obtained by the farmers. Farmers can also produce algae for countryard of the house [96]. Blue-green algal extracts comprise a great number of bioactive compounds that influence plant growth and development. They mostly contain growth phyto-regulators like gibberellins, auxin, cytokinin, ethylene and abscisic acid [100]. This group of microorganisms have been reported to benefit plants by producing growth promoting regulators resemble gibberellin and auxin, vitamins, amino acids, polypeptides, antibacterial and antifungal substances that exert phytopathogen biocontrol and polymers especially exopolysaccharides that were reported to enhance growth and productivity of plants like *Daucus carota* [101], *Santalum album* [102], *Oryzae sativa* [103], *Lilium alexandrae* hort [104] and *Beta vulgaris* L.[100]. Nonnitrogen fixing cyanobacteria can enrich phosphorus and potassium contents in soils, laying indirect major role in plant growth promotion [105]. Cyanobacteria also enhance the soil biological activity in terms of increasing the total bacterial, total cyanobacterial counts, CO2 evolution, dehydrogenase and nitrogenase activities. Many researches suggested that up to 50% of the recommended dose of the mineral nitrogen fertilizers could be saved by using some species of nitrogen fixing cyanobacteria. The obtained results emphasized the prospects and potentials of using cyanobacteria biofertilizers as renewable natural nitrogen resources for many crops. They are none polluting, inexpensive, utilize renewable resources (inorganic nutrients and atmospheric CO2) in addition to their ability in using free available

solar energy, atmospheric nitrogen and water [106-108].

**7.4. Mitigation of CO2: Why algae for CO2 sequestration?** 

Many options that have been proposed and that are in use for capturing CO2 can be seen as economically, socially and environmentally short-sighted. A common approach is taking measures to offset any immediate effects, often by simple relocation of the emissions. Injection of flue gases into oceanic or geological sinks is examples of such "end-of-pipe" solutions [109]. Algae cultivation can yield a broad range of useful end products, apart from biofuels. The sequestration of CO2 into algal biomass can become profitable also through the Wastewater nitrogen and phosphorous as microalgae nutrients aquaculture systems involving microalgae production and wastewater treatment (e.g. of amino acids, enzyme, or food industries wastewaters) seems to be quite promising for microalgae growth combined with biological cleaning. This allows nutrition of microalgae by using organic compounds (nitrogen and phosphorous) available in some manufactures wastewater, not containing heavy metals and radioisotopes. Additionally, microalgae can mitigate the effects of sewage effluent and industrial sources of nitrogenous waste such as those originating from water treatment or fish aquaculture and at the same time contributing to biodiversity. Moreover, removing nitrogen and carbon from water, microalgae can help reduce the eutrophication in the aquatic environment. Aslan and Kapdan [114] used *C. vulgaris* for nitrogen and phosphorus removal from wastewater with an average removal efficiency of 72% for nitrogen and 28% for phosphorus (from 3 to 8 mg/L NH4+ and 1.5–3.5 mg/L PO4 \_3). Other widely used microalgae cultures for nutrient removal are *Chlorella* [115] and *Spirulina* species [116]. Nutrient removal capacities of *Nannochloris* [117]), *Botryococcus brauinii* [118] and cyanobacterium *Phormidium bohneri* have also been investigated [119-120]. Environmental applications production of biodiesel and other bio-products from microalgae can be more environmentally sustainable, cost-effective and profitable, if combined with processes such as wastewater and flue gas treatments. In fact various studies demonstrated the use of microalgae for production of valuable products combined with environmental applications [113,121].

Microalgal Biotechnology: Prospects and Applications 301

technologies including production system development, algae harvest, algae strain development and genetic modification, algae products development, etc. Screening and genetic modification of algae strains will play an increasingly important role. Genetic engineering has the potential to improve the overall algal biomass yield and lipid yield. Discovery of new strains and genetically modified strains capable of secreting hydrocarbons to extracellular spaces will open some new opportunities; however, challenges with recovering the secreted liquids or volatiles remain. There is a need to develop high throughput screening and analysis methods. Current harvest and dewatering are still too energy intensive. New techniques and strategies must be devised to lower the costs. Direct conversions such as in situ transesterification and hydrothermal liquefaction offer the possibility to process wet algae. Fractionation of algal biomass, before or after oil extraction, deserves a closer look because it may play an important role in offsetting the costs. New techniques to disrupt algae cellular structures to improve oil extraction efficiency are needed

Metals are directly or indirectly involved in all phases of microbial growth. Many metals such as sodium, potassium, iron, copper, magnesium, calcium, manganese, zinc, nickel and cobalt are vital for biological functions, while others such as aluminum, cadmium, silver, gold, mercury and lead are not known to have necessary biological functions. All these elements can interact with microbial cells and be accumulated as a result of different mechanisms [127]. Some of these mechanisms have biotechnological importance and can be applied for the bioremediation of metals from industrial effluents. The capability of some microbial species to adsorb some heavy metals on their surface [128-129] or accumulate them within their structure is a chief route for the removal of heavy metals from contaminated environment [130-132]. Another fashion for the detoxification of heavy metals by microorganisms is the chelation of these metals inside or outside their cells after converting them into other forms to reduce their toxicity. In 2007, Lefebvre et al. [133] working with some cyanobacterial strains (*Limnothrix planctonica*, *Synechococcus leopoldiensis* and *Phormidium limnetica*) demonstrated their ability to convert Hg2+ into elemental mercury Hg° and meta-cinnabar (β-HgS) under pH controlled and aerated conditions. The transformation of mercury into β-HgS was attributed to the interaction with metal binding sulfhydryl protein as an intermediate step in metal sulfide synthesis. Moreover, some of the freshwater algae *Limnothrix planctonica* and *Selenastrum minutum* were recorded for their ability to bio-transform Hg2+ into a form with the analytical properties of β-HgS under aerobic conditions due to the presence of some protein and non-protein thiol chelators [134]. Furthermore, Lengke et al. [135] investigated the gold bioaccumulation by cyanobacterium Plectonema boryanum from gold (III)-chloride solutions. They confirmed that the reduction mechanism of gold (III) to metallic gold by this organism involves the formation of an intermediate gold (I)-sulfide due to a chelation process via some thiol compounds. Recntly Essa and Mostafa [136] studied the effeicincy of three cyanobacterial isolates (*Spirulina platensis*, *Nostoc muscorum*, and *Anabaena oryzae*) individually or as a mixed culture to

[126].

**7.7. Heavy metals and phycoremediation** 

#### **7.6. Biofuel production**

Microalgae can potentially be employed for the production of biofuels in an economically effective and environmentally sustainable manner. The production of these biofuels can be coupled with fuel gas CO2 mitigation, wastewater treatment and the production of highvalue chemicals. The efficiency is low but there is much room for improvement. The use of microalgae is seen as, at least, a partial solution to climate change and energy problem [122]. Many microalgae are exceedingly rich in oil which can be converted to biodiesel using existing technology. More than 50% of their biomass as lipids, sometimes even up to 80%, and oil levels of 20-50% are quite common [123].

**Figure 10.** Integration of microalgal bioreactors into existing wastewater and power generation infrastructures. The overall process uses microalgae to capture industrially produced waste CO2 in photobioreactors, coupled with treatment of nutrients in wastewater. CO2 is converted into algal biomass by photosynthesis in the presence of light. After processing (biological, physical or thermochemical), the biomass generated can be used for production of biodiesel, methane or other fuels and co-products (e.g. animal feeds and polymers)

Lipids production and biodiesel extraction from algae depend on algal species and extraction solvent system [124].There is a unique opportunity to both treat wastewater and provide nutrients to algae using nutrient-rich effluent streams. By cultivating microalgae, which consume polluting nutrients in municipal wastewater, and abstracting and processing this resource, then the goals of sustainable fuel production and wastewater treatment can be combined [174,125].The efforts span over many areas of "algae to fuels" technologies including production system development, algae harvest, algae strain development and genetic modification, algae products development, etc. Screening and genetic modification of algae strains will play an increasingly important role. Genetic engineering has the potential to improve the overall algal biomass yield and lipid yield. Discovery of new strains and genetically modified strains capable of secreting hydrocarbons to extracellular spaces will open some new opportunities; however, challenges with recovering the secreted liquids or volatiles remain. There is a need to develop high throughput screening and analysis methods. Current harvest and dewatering are still too energy intensive. New techniques and strategies must be devised to lower the costs. Direct conversions such as in situ transesterification and hydrothermal liquefaction offer the possibility to process wet algae. Fractionation of algal biomass, before or after oil extraction, deserves a closer look because it may play an important role in offsetting the costs. New techniques to disrupt algae cellular structures to improve oil extraction efficiency are needed [126].

#### **7.7. Heavy metals and phycoremediation**

300 Plant Science

applications [113,121].

**7.6. Biofuel production** 

and oil levels of 20-50% are quite common [123].

and co-products (e.g. animal feeds and polymers)

the use of microalgae for production of valuable products combined with environmental

Microalgae can potentially be employed for the production of biofuels in an economically effective and environmentally sustainable manner. The production of these biofuels can be coupled with fuel gas CO2 mitigation, wastewater treatment and the production of highvalue chemicals. The efficiency is low but there is much room for improvement. The use of microalgae is seen as, at least, a partial solution to climate change and energy problem [122]. Many microalgae are exceedingly rich in oil which can be converted to biodiesel using existing technology. More than 50% of their biomass as lipids, sometimes even up to 80%,

**Figure 10.** Integration of microalgal bioreactors into existing wastewater and power generation infrastructures. The overall process uses microalgae to capture industrially produced waste CO2 in photobioreactors, coupled with treatment of nutrients in wastewater. CO2 is converted into algal biomass by photosynthesis in the presence of light. After processing (biological, physical or

thermochemical), the biomass generated can be used for production of biodiesel, methane or other fuels

Lipids production and biodiesel extraction from algae depend on algal species and extraction solvent system [124].There is a unique opportunity to both treat wastewater and provide nutrients to algae using nutrient-rich effluent streams. By cultivating microalgae, which consume polluting nutrients in municipal wastewater, and abstracting and processing this resource, then the goals of sustainable fuel production and wastewater treatment can be combined [174,125].The efforts span over many areas of "algae to fuels" Metals are directly or indirectly involved in all phases of microbial growth. Many metals such as sodium, potassium, iron, copper, magnesium, calcium, manganese, zinc, nickel and cobalt are vital for biological functions, while others such as aluminum, cadmium, silver, gold, mercury and lead are not known to have necessary biological functions. All these elements can interact with microbial cells and be accumulated as a result of different mechanisms [127]. Some of these mechanisms have biotechnological importance and can be applied for the bioremediation of metals from industrial effluents. The capability of some microbial species to adsorb some heavy metals on their surface [128-129] or accumulate them within their structure is a chief route for the removal of heavy metals from contaminated environment [130-132]. Another fashion for the detoxification of heavy metals by microorganisms is the chelation of these metals inside or outside their cells after converting them into other forms to reduce their toxicity. In 2007, Lefebvre et al. [133] working with some cyanobacterial strains (*Limnothrix planctonica*, *Synechococcus leopoldiensis* and *Phormidium limnetica*) demonstrated their ability to convert Hg2+ into elemental mercury Hg° and meta-cinnabar (β-HgS) under pH controlled and aerated conditions. The transformation of mercury into β-HgS was attributed to the interaction with metal binding sulfhydryl protein as an intermediate step in metal sulfide synthesis. Moreover, some of the freshwater algae *Limnothrix planctonica* and *Selenastrum minutum* were recorded for their ability to bio-transform Hg2+ into a form with the analytical properties of β-HgS under aerobic conditions due to the presence of some protein and non-protein thiol chelators [134]. Furthermore, Lengke et al. [135] investigated the gold bioaccumulation by cyanobacterium Plectonema boryanum from gold (III)-chloride solutions. They confirmed that the reduction mechanism of gold (III) to metallic gold by this organism involves the formation of an intermediate gold (I)-sulfide due to a chelation process via some thiol compounds. Recntly Essa and Mostafa [136] studied the effeicincy of three cyanobacterial isolates (*Spirulina platensis*, *Nostoc muscorum*, and *Anabaena oryzae*) individually or as a mixed culture to precipitate some heavy metals (Hg2+, Cd2+, Cu2+ and Pb2+) out of their solutions through using the culture biogas produced during their aerobic growth in a batch bioreactor. Variable capabilities of metal bioprecipitation were recorded by the three algal isolates. FT-IR studies showed the existence of –OH groups in the metal precipitate produced by the algal isolates while –NH groups were identified only in the metal precipitates produced by *N. muscorum*, and *A. oryzae*. This study highlighted a novel approach for heavy metals bioremediation through the transformation of these metals into nitrogen complexes and/or hydroxide complexes via using the culture biogas produced by some cyanobacterial species.

Microalgal Biotechnology: Prospects and Applications 303

*Chlorella* are in excess of US\$ 38 billion [85]. The most important substance in *Chlorella* is β-1,3-glucan, which is an active immunostimulator, a free-radical scavenger and a reducer of blood lipids (9, Ryll et al., Abstr. Europ. Workshop Microalgal Biotechnol., Germany, p. 56, 2003). However, various other health-promoting effects have been clarified (efficacy on gastric ulcers, wounds, and constipation; preventive action against atherosclerosis and

hypercholesterolemia; and antitumor action) [85,122,138].

**Figure 11.** Earthrise Farms *Arthrospira* production plant (Calipatria, CA, USA)

**Figure 12.** Earthrise Farms microalgal production process

#### **8. Microalgal production**

Microalgae for human nutrition are nowadays marketed in different forms such as tablets, capsules and liquids. They can also be incorporated into pastas, snack foods, candy bars or gums, and beverages. In addition, this microalga has various possible healthpromoting effects: the alleviation of hyperlipidemia, suppression of hypertension, protection against renal failure, growth promotion of intestinal Lactobacillus, and suppression of elevated serum glucose level [86-87]. Owing to their diverse chemical properties, they can act as a nutritional supplement or represent a source of natural food colorants. The commercial applications are dominated by four strains: *Arthrospira, Chlorella, D. salina* and *Aphanizomenon flos-aquae*. *Arthrospira* is used in human nutrition because of its high protein content and its excellent nutritive value [87,88,137]. A significant amount of *Arthrospira* production is realized in China and India. The world's largest producer Hainan Simai Enterprising Ltd. is located in the Hainan province of China. This company has an annual production of 200 t of algal powder, which accounts for 25% of the total national output and almost 10% of the world output. The largest plant in the world is owned by Earthrise Farms and streches over an area of 440,000 m2 (located at Calipatria, CA, USA; Figure 11). Their production process is presented in Figure 12. Their Arthrospira-based products (tablets and powder) are distributed in over 20 countries around the world. Many other companies sell a wide variety of nutraceuticals made from this microalga. For example, the Myanmar *Spirulina* Factory (Yangon, Myanmar) sells tablets, chips, pasta and liquid extract, and Cyanotech Corp. (a plant in Kona, Hawaii, USA) produces products ranging from pure powder to packaged bottles under the name *Spirulina pacifica*. Cyanotech Corp. has developed an orginal process for drying the biomass in order to avoid the oxidation of carotenes and fatty acids that occurs with the use of standard dryers. The patented process employs a closed drying system that is kept at low oxygen concentrations by flushing with nitrogen and carbon dioxide. The process relies on a very cold ocean water crown from a depht of 600 m just offshore to provide dehumidification and actually dries microalgal products in less than 6 s (Figure 13). *Chlorella* is produced by more than 70 companies; Taiwan Chlorella Manufacturing and Co. (Taipei, Taiwan) is the largest producer with 400 t of dried biomass produced per year. Significant production is also achieved in Klötze, Germany (130 – 150 t dry biomass per year) with a tubular photobioreactor. This reactor consists of compact and vertically arranged horizontal running glass tubes with a total length of 500,000 m and a total volume of 700 m3 (Figure 14). The world annual sales of *Chlorella* are in excess of US\$ 38 billion [85]. The most important substance in *Chlorella* is β-1,3-glucan, which is an active immunostimulator, a free-radical scavenger and a reducer of blood lipids (9, Ryll et al., Abstr. Europ. Workshop Microalgal Biotechnol., Germany, p. 56, 2003). However, various other health-promoting effects have been clarified (efficacy on gastric ulcers, wounds, and constipation; preventive action against atherosclerosis and hypercholesterolemia; and antitumor action) [85,122,138].

302 Plant Science

**8. Microalgal production** 

precipitate some heavy metals (Hg2+, Cd2+, Cu2+ and Pb2+) out of their solutions through using the culture biogas produced during their aerobic growth in a batch bioreactor. Variable capabilities of metal bioprecipitation were recorded by the three algal isolates. FT-IR studies showed the existence of –OH groups in the metal precipitate produced by the algal isolates while –NH groups were identified only in the metal precipitates produced by *N. muscorum*, and *A. oryzae*. This study highlighted a novel approach for heavy metals bioremediation through the transformation of these metals into nitrogen complexes and/or hydroxide complexes via using the culture biogas produced by some cyanobacterial species.

Microalgae for human nutrition are nowadays marketed in different forms such as tablets, capsules and liquids. They can also be incorporated into pastas, snack foods, candy bars or gums, and beverages. In addition, this microalga has various possible healthpromoting effects: the alleviation of hyperlipidemia, suppression of hypertension, protection against renal failure, growth promotion of intestinal Lactobacillus, and suppression of elevated serum glucose level [86-87]. Owing to their diverse chemical properties, they can act as a nutritional supplement or represent a source of natural food colorants. The commercial applications are dominated by four strains: *Arthrospira, Chlorella, D. salina* and *Aphanizomenon flos-aquae*. *Arthrospira* is used in human nutrition because of its high protein content and its excellent nutritive value [87,88,137]. A significant amount of *Arthrospira* production is realized in China and India. The world's largest producer Hainan Simai Enterprising Ltd. is located in the Hainan province of China. This company has an annual production of 200 t of algal powder, which accounts for 25% of the total national output and almost 10% of the world output. The largest plant in the world is owned by Earthrise Farms and streches over an area of 440,000 m2 (located at Calipatria, CA, USA; Figure 11). Their production process is presented in Figure 12. Their Arthrospira-based products (tablets and powder) are distributed in over 20 countries around the world. Many other companies sell a wide variety of nutraceuticals made from this microalga. For example, the Myanmar *Spirulina* Factory (Yangon, Myanmar) sells tablets, chips, pasta and liquid extract, and Cyanotech Corp. (a plant in Kona, Hawaii, USA) produces products ranging from pure powder to packaged bottles under the name *Spirulina pacifica*. Cyanotech Corp. has developed an orginal process for drying the biomass in order to avoid the oxidation of carotenes and fatty acids that occurs with the use of standard dryers. The patented process employs a closed drying system that is kept at low oxygen concentrations by flushing with nitrogen and carbon dioxide. The process relies on a very cold ocean water crown from a depht of 600 m just offshore to provide dehumidification and actually dries microalgal products in less than 6 s (Figure 13). *Chlorella* is produced by more than 70 companies; Taiwan Chlorella Manufacturing and Co. (Taipei, Taiwan) is the largest producer with 400 t of dried biomass produced per year. Significant production is also achieved in Klötze, Germany (130 – 150 t dry biomass per year) with a tubular photobioreactor. This reactor consists of compact and vertically arranged horizontal running glass tubes with a total length of 500,000 m and a total volume of 700 m3 (Figure 14). The world annual sales of

**Figure 11.** Earthrise Farms *Arthrospira* production plant (Calipatria, CA, USA)

**Figure 12.** Earthrise Farms microalgal production process

Microalgal Biotechnology: Prospects and Applications 305

introduced in microalgal biotechnology to produce vitamins, proteins, cosmetics, health foods and animal feed. For most of these applications, the market is still developing and the biotechnological use of microalgae will extend into new areas.With the development of algal cultures and screening techniques, microalgal biotechnology can meet the challenging demands of food, feed, pharmaceutical industries, fuels and biofertilizers. The general needs of the human society are continuously increasing. We need every new compound which may be useful for the human society. More food, new drugs, and other goods are highly necessary for the benefit of humankind. The only question is the existence of sufficient natural and technical resources to fulfill these demands. Fortunately, in the area of the research of bioactive microbial products it seems that the ever expanding scientific and technical possibilities are increasing together with the continuously widening needs of the

*Microbiology Department, Soil, Water and Environment Research Institute, Agricultural Research* 

[1] Pratt DE (1992) Natural antioxidants from plant material. In: Huang MT, Ho CT and Lee CY (Editors), Phenolic Compounds in Food and their Effects on Health. II.

[3] Li HB, Cheng KW, Wong CC, Fan KW, Chen F and Jiang Y (2007) Evaluation of antioxidant capacity and total phenolic content of different fractions of selected

[4] Richmond A. (2004) Handbook of microalgal culture: biotechnology and applied

[5] Brennan L and Owened PAuthor Vitae (2010) Biofuels from microalgae-Areview of technologies for production, processing and extractions of biofuels and co-products.

[6] Dos Santos MD, Guaratini T, Lopes JLC, Colepicolo P and Lopes NP (2005) Plant cell and microalgae culture. In:Modern Biotechnology in Medicinal Chemistry and

[7] Burja AM, Banaigs B, Abou-Mansour E, Burgess JG and Wright PC (2001) Marine cyanobacteria - a prolific source of natural products. Tetrahedron. 57:9347-9377. [8] Tyagi S, Singh G, Sharma A and Aggarwal G (2010) Phytochemicals as candidate therapeutics: An over view. International Journal of Pharmaceutical Sciences Review

American Chemical Society. ACS Symposium Series, 507.Washington. 54-71. [2] Abou El alla FM and Shalaby EA (2009) Antioxidant Activity of Extract and Semi-Purified Fractions of Marine Red Macroalga. Gracilaria Verrucosa. Aust J. Bas. App. Sci.

human.

**Author details** 

Soha S.M. Mostafa

**10. References** 

3:3179-85.

*Center, Giza, Egypt Acknowledgement* 

microalgae. Food Chemistry. 102:771–776.

Industry. Kerala, India: Research Signpost.

Renewable and Sustainable Energy Reviews. 14(2):557-577.

phycology. Blackwell Science Ltd.

and Research. 3(1):53-55.

**Figure 13.** Cyanotech process for drying microalgae biomass

**Figure 14.** Glass tube photobioreactor (700 m3) producing Chlorella biomass (Klötze, Germany)

## **9. Conclusion**

Meeting the increasing water demands with limited resources advocates Egypt to find innovative and sustainable approaches for management. It is essential to maximize the benefits of the available resources and to minimize the wastes and losses, not only in water resources but also in all economical and social resources, and in an integrated framework believing that everything is related to everything. So would it not be possible to kill several birds with one stone, using algae for absorbing CO2 at the same time as providing nutrient recovery from food industrial effluents and domestic wastewater and producing renewable energy (fuels), as well as other pharmaceutical products, food, feed and fertilizer from the biomass? In recent years, microalgal culture technology is a business oriented line owing to their different practical applications. Innovative processes and products have been introduced in microalgal biotechnology to produce vitamins, proteins, cosmetics, health foods and animal feed. For most of these applications, the market is still developing and the biotechnological use of microalgae will extend into new areas.With the development of algal cultures and screening techniques, microalgal biotechnology can meet the challenging demands of food, feed, pharmaceutical industries, fuels and biofertilizers. The general needs of the human society are continuously increasing. We need every new compound which may be useful for the human society. More food, new drugs, and other goods are highly necessary for the benefit of humankind. The only question is the existence of sufficient natural and technical resources to fulfill these demands. Fortunately, in the area of the research of bioactive microbial products it seems that the ever expanding scientific and technical possibilities are increasing together with the continuously widening needs of the human.

#### **Author details**

304 Plant Science

**9. Conclusion** 

**Figure 13.** Cyanotech process for drying microalgae biomass

**Figure 14.** Glass tube photobioreactor (700 m3) producing Chlorella biomass (Klötze, Germany)

Meeting the increasing water demands with limited resources advocates Egypt to find innovative and sustainable approaches for management. It is essential to maximize the benefits of the available resources and to minimize the wastes and losses, not only in water resources but also in all economical and social resources, and in an integrated framework believing that everything is related to everything. So would it not be possible to kill several birds with one stone, using algae for absorbing CO2 at the same time as providing nutrient recovery from food industrial effluents and domestic wastewater and producing renewable energy (fuels), as well as other pharmaceutical products, food, feed and fertilizer from the biomass? In recent years, microalgal culture technology is a business oriented line owing to their different practical applications. Innovative processes and products have been Soha S.M. Mostafa

*Microbiology Department, Soil, Water and Environment Research Institute, Agricultural Research Center, Giza, Egypt Acknowledgement* 

#### **10. References**


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## *Edited by Nabin Kumar Dhal and Sudam Charan Sahu*

The book "Plant Science" consists of 12 chapters divided into three sections authored by many researchers from different parts of the Globe. Section-I: Plant and Environment, describes the relationship between plants and environment, particularly enumerating species-environment relationship and response of plants to different environmental stress conditions. Section-II: Plant-Microbe relation, embodies broadly on both positive and negative aspects of microbes on plants. Section-III: Plant Biotechnology, shed light on current biotechnological research to develop modern technology for producing biologicals and also increasing plant immunity in present environmental conditions. The book "Plant Science" will be helpful to a wide group peoples; readers, scientists, researchers and allied professionals. We recommend it to you; enjoy reading it, save the plant and save life!

Plant Science

Plant Science

*Edited by Nabin Kumar Dhal* 

*and Sudam Charan Sahu*