Strategic role of nanotechnology for fertilizers: potential and limitations

The ability of people to construct and manipulate materials at a nano-scale has increased tremendously during the last decade, building the fundamentals of the interdisciplinary science nanotechnology. Nanomaterials behave differently than the same material at a non-nano scale; they have high surface area to volume ratio, high solubility, and specific targeting due to small size, high mobility, and low toxicity. They can be engineered for surface reactivity or other desired characteristics - unique behaviour that can be both useful and profitable. As of March 2011, over 1300 commercially available products contain nanomaterials. Nanotechnology was a $1 trillion industry in 2015.

According to the National Nanotechnology Initiative (NNI) (https://www.nano.gov/about-nni ), “Nanotechnology research and development is directed towards understanding and creating improved materials, devices and systems that exploit nanoscale properties”. Following the definition of the Royal Society, "Nanotechnologies are the design, characterization, production and application of structures, devices and systems by controlling shape and size at nanometer scale".

Recently, nanotechnology has emerged as the sixth revolutionary technology after the green revolution of the 1960s and the biotechnology revolution of the 1990s. Nanotechnology is a novel scientific approach that involves the use of materials and equipment capable of manipulating physical and chemical properties of a substance at molecular levels. It merges science and technology, leading to revolutionary breakthrough in electronics, energy, remediation, automobile, space technology, and life sciences. The potential uses and benefits of nanotechnology are enormous. Nowadays, nanotechnology is progressively moved away from the experimental into the practical areas. Among others, it promises significant contribution to agricultural research in solving important agricultural problems, such as detection of pollutants, plant diseases, pests, and pathogens; controlled delivery of pesticides, fertilizers, nutrients, and genetic material; formation and binding of soil structure. Today, when agricultural scientists are facing major challenges such as reduced crop production, nutrient deficiency and climate change, nanotechnology has offered promising applications for precision farming. This innovative technology embraces wide applications such as plant disease control, enhanced nutrient uptake, improved plant growth, and sustained release of agrochemicals. Interestingly, a nanoparticle (NP)-based strategy has gained momentum and has become increasingly popular in the agricultural sector as a result of its unique properties compared with those of the biopesticides. The application of nanotechnology to agriculture (the so-called agri-nanotechnology, Fig. 1) is getting significant attention, primary in the following several categories:

• Increase production rates and yield;
• Increase efficiency of resource utilization;
• Minimize waste production;
• Specific applications that include nano-fertilizers and nano-pesticides;
• Nano-based treatment of agricultural waste;
• Nano-sensors.

Fig. 1. Multidisciplinary nature of agri-nanotechnology.

Currently, the potential of nanotechnology in sustainable agriculture management is clearly recognized. It occupies a prominent position in transforming agriculture and food production. The development of nano-devices and nanomaterials could forward novel applications in plant biotechnology and agriculture. Thus, the development of slow/controlled release fertilizers on the basis of nanotechnology has now become crucial for promoting the development of environmentally friendly and sustainable agriculture. Applying nanoscale or nanostructured materials as fertilizer carriers leads to the development of the so-called “smart fertilizer” – new facilities that enhance the nutrient use efficiency and reduce the costs of environmental protection.

Nano-fertilizers vs. conventional fertilizers: formulation and delivery of nano-fertilizers

The outburst of world population in the last 10–15 years has imposed the necessity for higher agriculture productivity to satisfy the food needs of billions of people. The increasing nutrient deficiency in soils causes significant economic losses for farmers on the one hand and considerable decreases in nutritional quality of grain for food and feed. The crop productivity can be enhanced through application of fertilizers, although they have an additional role in enhancing the food production especially after the introduction of high yielding and fertilizer responsive crop varieties. Conventional fertilizers are generally applied on the crops by either spraying or broadcasting. An important factor, on which the mode of application depends, is the real final concentration of the fertilizers in the plants. Conventional fertilizers offer nutrients in chemical forms that are not fully accessible to plants. Additionally, the inversion of these chemicals to insoluble form in soil is the reason for the very low utilization of most of the macronutrients. A concentration much below the minimal desired one reaches to the targeted site due to leaching of chemicals, drift, runoff, evaporation, hydrolysis by soil moisture, and photolytic and microbial degradation. It has been estimated that around 40–70 % of nitrogen, 80–90 % of phosphorus, and 50–90 % of potassium content of applied fertilizers are lost in the environment and never reach the plant. These problems superimpose repeated use of fertilizers. According to the International Fertilizer Industry Association, world fertilizer consumption sharply picked up in 2009–2010 and 2010–2011 with growth rates of 5–6 %. World demand is estimated to reach 192.8 Mt by 2016–2017. The repeated use, in turn, adversely affects the inherent nutrient balance of the soil and results in environmental pollution affecting normal flora and fauna. It is reported that excess use of fertilizers increases pathogen and pest resistance, reduces soil microflora, diminishes nitrogen fixation, contributes to bioaccumulation of pesticides, and destroys habitats for birds. This vicious circle causes sustainable and economic losses.

It is well known that the yields of many crops have begun to drop as a result of imbalanced fertilization and decrease in soil organic matter. Moreover, excessive applications of nitrogen and phosphorus fertilizers affect the groundwater and also lead to eutrophication in aquatic ecosystems. The remaining minerals may either leach down and/or leak and become fixed in the soil, or contribute to air pollution. Considering these facts, the large-scale application of chemical fertilizers to increase the crop productivity is not an acceptable option for sustainability. Especially in a long-term perspective, although the conventional fertilizers increase the crop production, they disturb the soil mineral balance and decrease the soil fertility. In addition to the irreparable damage that the excess use of chemical fertilizers causes to the soil structure and mineral cycles, it spoils the soil microflora, plants, and consequently, the food chains across ecosystems, leading to heritable mutations in future generations of consumers. Thus, there is an urgent need to optimize the use of chemical fertilization to fulfil the crop nutrient requirements and to minimize the risk of environmental pollution. Accordingly, it is very important to develop smart materials that can systematically release chemicals to specific targeted sites in plants which could be beneficial in controlling the nutrition deficiency in agriculture, while keeping the natural soil structure and contributing to clean environment. Nano-fertilizers are promising alternative in this context.

A nano-fertilizer refers to a product in nanometer scale that delivers nutrients to crops. Nano-fertilizer technology is a recent innovation. Substituting traditional methods of fertilizer application by nano-fertilizers is an approach to release nutrients into the soil both gradually and in a controlled way. Nano-fertilizers show controlled release of agrochemicals through site-targeted delivery, reduction in toxicity, and enhanced nutrient utilization of delivered fertilizers. They possess unique features that enhance plants’ performance in terms of ultrahigh absorption, increase in production, rise in photosynthesis, and significant expansion in the leaves’ surface area. Besides, the controlled release of nutrients contributes to preventing eutrophication and pollution of water resources.

In nano-fertilizers, nutrients can be encapsulated by nanomaterials, coated with a thin protective film, or delivered as emulsions or nanoparticles. There are many throughput examples of nano-fertilizers application. Thus, treatment with TiO₂ nanoparticles on maize had a considerable effect on growth, whereas the effect of TiO₂ bulk treatment was negligible. Titanium nanoparticles increased the light absorption and photo energy transmission. In another experiment, a compound of SiO₂ and TiO₂ nanoparticles increased the activity of nitrate reductase in soybeans and intensified the plant absorption capacity, making the use of water and fertilizer more efficient. A nano-organic iron-chelated fertilizer is proved to be environmentally sustainable. The positive effect from the uptake and penetration of ZnO₂ nanoparticles on tomato plants leaves supports its potential use as a future nano-fertilizer. Nano-fertilizers that ensure slow, targeted, efficient release have the potential to increase the efficiency of nutrient uptake. Engineered nano-particles are useful for mitigating the chronic problem of moisture retention in arid soils and enhancing crop production by increasing the availability of nutrients in the rhizosphere. Coating and binding of nano-particles help to regulate the release of nutrients from the fertilizer capsule. Application of a nano-composite consisting of nitrogen, phosphorus, potassium, micronutrients, mannose, and amino acids enhanced the uptake and use of nutrients by grain crops. Zn–Al layered double-hydroxide nano-composites have been employed for the controlled release of chemical compounds that act as plant growth regulators. Nano-porous zeolite based on nitrogen fertilizer can be used as an alternate strategy to improve the efficiency of nitrogen use in crop production systems. As super-fertilizer, carbon nanotubes were found to penetrate tomato seeds and affect their germination and growth rates. Analytical methods indicated that the carbon nanotubes penetrated the thick seed coat and supported water uptake inside seeds.

These facts support the statement that fertilizers based on nanotechnology have the potential to surpass conventional fertilizers following several important indices (as shown in Table 1).

Table 1. Conventional fertilizers vs. nano-fertilizers

Index	Nano-fertilizer	Conventional fertilizer
Solubility	High	Low
Dispersion of mineral micronutrients	Improved dispersion of insoluble nutrients	Lower solubility due to large particle size
Soil adsorption and fixation	Reduced	High
Bioavailability	High	Low
Efficiency of nutrients uptake	Increased uptake ratio; saves fertilizer resource	Conventional fertilizer is not available to roots and the nutrients uptake efficiency is low
Controlled release	Release rate and pattern precisely controlled	Excess release leading to toxicity and soil imbalance
Effective duration of release	Extended effective duration	Used by the plant at the site and time of application; the rest is converted into insoluble form
Loss rate	Reduced loss of fertilizer nutrients	High loss rate due to leaching, drifting, run-off

The nano-fertilizers should be formulated in a way that they retain important properties such as high solubility, stability, effectiveness, time-controlled release, enhanced targeted activity with effective concentration, and less eco-toxicity due to the safe, easy mode of delivery and disposal.

Nanoparticles possess a great potential in targeted delivery of nutrients to living systems. Nanoparticles can be loaded by nutrients most commonly through one of the following ways:

• absorption on the nanoparticles;
• attachment on the nanoparticles mediated by ligands;
• encapsulation in nanoparticulate polymeric shell;
• entrapment in nanoparticles.

Thus, it has been shown that chitosan nanoparticles suspensions containing N, P, and K fertilizers can be useful for agricultural applications. Similarly, urea-modified hydroxyapatite (HA) nanoparticles are exploited for slow and sustained release of nitrogen over time with the crop growth. The large surface area of HA facilitates the large amount of urea attachment on the HA surface and the strong interaction between HA nanoparticles and urea contributes to the slow and controlled release of urea. Polymer-based mesoporous nanoparticles can also provide an efficient carrier system to agrochemical compounds. Mesoporous silica nanoparticles (150 nm) have been reported to entrap urea and to release it in a controlled manner in soil and water.

The efficiency of nano-fertilizers and their impact on plant systems is influenced by the method of their application. The delivery of nano-fertilizers to plants can be realized through the methods listed below. The approaches include either in vitro or in vivo application, as shown in Table 2.

Table 2. Modes of nano-fertilizer application

In vitro methods	In vivo methods
Aeroponics:	Soil Application:
Hydroponics:	Foliar Application

• Principle: the technique, first reported in 1992, consists in continuously spraying of a nutrient solution on roots suspended in air.
• Advantages: the technique allows strict control of the gaseous environment around the roots.
• Disadvantages: the technique requires a high level of nutrients to sustain rapid plant growth, thus its application is restricted.
• Principle: direct delivery to soli;
• Requirements: careful choose of the persistence time of the fertilizer in the soil; special attention to the soil texture, salinity, plant sensitivity to salts, and pH of the amendment. Negative soil particles affect the adsorption of mineral nutrients. The anion exchange capacity of most agricultural soils is small compared to the cation one. Among anions, NO_3- remains mobile in the soil solution and is susceptible to leaching by water, PO₄^3- binds to soil particles containing Al or Fe because the positively charged Fe^2+/3+ and Al³⁺ exchange OH- groups with phosphates, resulting in tight bounding of the latter, whose mobility and availability in soil can limit plant growth.
• Advantages: the most common method of nutrient supplementation using chemical and organic fertilizers.
• Principle: the plants are grown with their roots immersed in a liquid nutrient solution (without soil), introduced in 1937 for dissolved inorganic salts, also known as the so-called “solution culture”.
• Requirements: careful choice of the volumes of nutrient solution, maintenance of oxygen demands and pH.
• Advantages: application of supporting materials (e.g. sand) that allow the nutrient solution to be flushed from one end and old solution to be removed from the other end.
• Disadvantages: frequent pathogen attack and high moisture rates which may cause overwilting of soil-based plants.
• Principle: liquid fertilizers are directly sprayed onto leaves, generally used for the supply of trace elements.
• Advantages: reduces the time lag between application and uptake by plant during the rapid growth phase; circumvents the problem of restricted uptake of a nutrient from soil; agronomic advantage of foliar application, since stomata and leaf epidermal cells are majorly involved in nutrient uptake.
• Disadvantages: further needs for standardization of an application protocol to avoid damage to the leaves; need of specific time (morning and evening) of spraying because the stomata open during these time periods only; possibility of plant damage if incorrect concentration of fertilizer is applied.

Technology expansion has improved the ways for large-scale production of nanoparticles of physiologically important metals, which are now used as “smart delivery systems” in order to improve fertilizer formulation by minimizing nutrient loss and increasing the uptake in plant cells. “Smart delivery systems” means a combination of specifically targeted, highly controlled, remotely regulated, and multifunctional characteristic to avoid biological barriers for successful targeting. The specific properties of nano-fertilizers, i.e. their high surface area, sorption capacity, and controlled-release kinetics to targeted sites, attribute them as a smart delivery system.

Smart fertilizers are becoming a reality through transformed formulation of conventional products using nanotechnology. The nanostructured formulation allows a fertilizer to intelligently control the release speed of nutrients in order to match the uptake pattern of a specific crop. It improves the solubility and dispersion of insoluble nutrients in the soil, reduces the soil absorption and fixation and increases the bioavailability and, hence, the nutrient uptake efficiency.

Biosynthesis of nanoparticles by microorganisms

Mediated synthesis of metal nanoparticles by microorganisms

In recent years, the use of biological entities has emerged as a novel method for the synthesis of nanoparticles. The biotechnological synthesis of nanoparticles has many advantages, such as the use of known microbial technologies and processes to scale up the obtaining of biomass. This is leading to economic viability, possibility of readily covering large surface areas by suitable growth of microbes, which is of major advantage in the field of agriculture for easier production of bio-fertilizers.

The disadvantages of the conventional methods for obtaining metal nanoparticles like high energy demands and high fabrication cost, as well as toxic by-products production, makes the implementation of such approaches at large scale very complicated. The use of microbial cell factories like bacteria, fungi, algae, viruses and actinomycetes provides a smart alternative way of synthesis of metallic nanoparticles. The biosynthesis of metallic nanoparticles in such microorganisms is a low-cost and eco-friendly technology. The use of a broad number of microorganisms, prokaryotic as well as eukaryotic ones, takes part in the synthesis of a wide range of metal nanoparticles, such as gold (Au), silver (Ag), lead (Pb), platinum (Pt), copper (Cu), iron (Fe), cadmium (Cd) and metal oxides such as titanium oxide (TiO), zinc oxide (ZnO), etc. These microorganisms provide varied conditions for the production of nanoparticles. The nanoparticles produced are highly useful, safe and environmentally friendly in nature with a lot of applications ((Syed, PhD Thesis). In agriculture, the nanoparticles most widely used as bioeffectors are coper (Cu), iron (Fe), silver (Ag) and gold (Au). The future challenges in this respect comprise optimal biosynthesis of nanoparticles with defined size and shape as well as optimal duration of the fermentation process in order to enhance their stability.

Microbiological synthesis is a new approach to the manufacture of nanoparticles and realization of the so-called bio-nanofactories. The major characteristics of nanoparticles have been revealed in research on prepared nanoparticles of desirable shape and size.

The principal flow chart for microbiological synthesis of metallic nanoparticles is presented in Fig. 2.

Fig.2 Principal flow chart for microbiological synthesis of metallic nanoparticles

The following important parameters play a significant role in the biosynthesis of nanoparticles.

1 Bioresources used for nanoparticle biosynthesis: The synthesis of nanoparticles is characterized by choice of the most convenient microorganism with respect to: growth rate, enzyme production and the respective metabolic pathways. Some microorganisms, like bacteria, viruses, fungi, yeasts and algae, are used for the biosynthesis of metallic nanoparticles and are an object of specific research.

2. Cellular metabolites involved in biosynthesis: Molecules like enzymes, proteins, polysaccharides etc. act as reducing and stabilizing agents in the biosynthesis of nanoparticles. They can be utilized in the process as whole-cell microorganisms, crude cell preparations, and crude or purified enzymes obtained from microorganisms. The obtained nanoparticles result mainly from bioreduction, which is realized by co-enzymes such as NADH, NADPH, FAD, etc. It has been found that nanoparticle synthesis with the help of whole fungal cells is much cheaper as compared to that using purified enzymes from the same fungal strain (Syed, PhD Thesis).

3. Reactions facilitating nanoparticle biosynthesis: The process of this biosynthesis is initiated with harvesting of microbial biomass, which is related with residual nutrients and metabolites to avoid undesired by-product reactions. During the processes of scalling up, the production rate and product yield are of special interest and optimization is necessary (e.g. production time, pH, temperature etc.). The process of optimization of these factors can influence the particles morphology and their properties. Thus, researchers have currently directed their investigations on arranging the optimal reaction conditions as well as the equipment used in the bioreduction process (Syed, PhD Thesis).

4 Growth of inoculum for biosynthesis of nanoparticles: The biosynthesis of nanoparticles depends on the growth conditions of the microbial producers, e.g. nutrients, pH, temperature, etc. These factors need to be optimized. They are also important in case of using whole cells and crude enzymes. Another important parameter for optimization of the inoculums is the harvesting time, so it is necessary to monitor the enzyme activities during the growth (Syed, PhD Thesis).

Microbial nanoformulations: exploring the potential for nano-farming

Nanoparticles synthesized by microbes are highly stable and could offer a non-toxic, cost-effective and eco-friendly approach for synthesis over chemical ones. This green synthesis has a great advantage over the chemical methods, which have toxic effects on the environment. Thus, the use of agriculturally important microorganisms for nanoparticle biosynthesis and their further role in agriculture is of considerable significance. The use of nanoformulations may enhance the stability of bio-fertilizers and bio-stimulators with respect to desiccation, heat, and UV inactivation.

Nano-fertilizers uptake, translocation, and fate in plants

The uptake and fate of nano-fertilizers in plants is an emerging field of research interest. The uptake, translocation, and accumulation of nanoparticles depend on the plant itself, more specifically, on the plant species, age, and growth environment. These processes are also linked to the physicochemical properties, functionalization, stability, and mode of delivery of the nanoparticles. A schematic representation of the uptake, translocation, and biotransformation pathway of various nanoparticles is proposed by Rico et al. (2011) along with possible modes of cellular uptake in the plant system. According to this presentation, the root system uptakes and translocates ZnO²⁺, Cu²⁺, Al³⁺, Ag²⁺ and Fe₃O₄ nanoparticles (NP) to the foliar part of a plant, regardless of its species. In addition, indicatives for species dependence are available for the translocation of Cu NP, ZnO NP, Al NP and Ag NP (all in leaves), Ni(OH)₂ NP in the stem, and CeO₂ NP in both the stem and leaves. Translocation of Fe₃O₄ NP in the stem is also speculated.

The probable differential interaction of nanoparticles on exposure in the root absorption zone is summarized in Table 3.

Table 3. Localization and interaction of different nanoparticles in the root absorption zone.

Nanoparticle	Localization / interaction
Fe3O4 NP	Cambium
ZnO NP	Endodermis, metaxylem; Zn2+ - in the metaxylem
CeO2 NP	Cortex
Al NP	Cortex Al3+ - in the metaxylem
Ag NP	Cortex; Ag2+ - in the metaxylem
Cu NP	Cortex; Cu2+ - in the cambium and metaxylem
TiO2 NP	Cortex
Ni (OH)2 NP	Metaxylem

The entry of the nanoparticles through the cell wall depends on the cell wall pore diameter (5–20 nm). That is why nanoparticles or nanoparticle aggregates with a diameter less than the pore size of the plant cell walls can easily enter through the cell wall and reach up to the plasma membrane. Functionalized nanoparticles can facilitate the enlargement of the pore size or the induction of new cell-wall pore formation to enhance the uptake of nanoparticles. Research discussions are going on about the uptake of nanoparticles into plant cells mediated by binding to carrier proteins through aquaporin, ion channels or endocytosis. Additionally, nanoparticles can also be transported into the plant by forming complexes with membrane transporter proteins or root exudates. Other studies report that nanoparticles could enter through the stomata or trichome bases in leaves. Studies on the uptake and translocation of TiO₂–alizarin red S complex in Arabidopsis thaliana seedlings have revealed that the mucilage released by the roots develops a pectin hydrogel complex around the root, which is most probably responsible for the entry of the nanoparticle–dye complex.

Recent studies on the mechanism of nanoparticle uptake and translocation have exploited fluorescently labeled monodispersed mesoporous silica nanoparticles which have been shown to penetrate the roots via the symplastic and apoplastic pathways and translocate via the xylem tissue to the aerial parts of plants, including the stem and leaves. However, the exact mechanism of nanoparticle uptake by plants is still not fully elucidated.

In the cytoplasm, nanoparticles are targeted to different cytoplasmic organelles and interfere with different metabolic processes of the cell (Table 3). It is shown that the uptake of TiO₂ nanoparticles in wheat includes localization in parenchyma and vascular tissues of the root. The cell internalization and upward translocation of ZnO nanoparticles in Lolium perenne (ryegrasses) proceeds through the root cells and then they move up to the vascular tissues.

The uptake and accumulation of ZnO nanoparticles, when applied at higher concentrations, is hindered, since the nanoparticles get agglomerated, which inhibits their entry through the cell-wall pores. Moreover, X-ray absorption spectroscopy of ZnO-treated seedlings revealed presence of Zn²⁺ ions instead of ZnO, suggesting a role of the roots in ZnO ionization on the surface.

Another class of nanoparticles, the magnetite NP, behave in a way that their presence in root, stem and leaves can be reported, and the extent of the nanoparticle uptake has been proved to be affected by the type of the growth medium. A higher uptake was achieved in hydroponic medium as compared to that observed in plants grown in sand, whereas no uptake was observed in plants grown in soil, which might be due to the adherence of magnetite nanoparticles to soil and sand grains.

Finally, it is noteworthy that, besides some conclusive studies on TiO₂ and ZnO nanoparticles, most of the uptake, translocation, and accumulation studies in plants are reported only up to the germination stage. Hence, the fate of nanoparticles in the plant system is still largely unknown.

Effect of nano-fertilizers on plant physiology and metabolism

Most recent studies support the idea that nanoparticles exert some adverse effects on plants. However, there are a few studies that have suggested that nanoparticles, when delivered in a controlled safe dose, may contribute to promotion of plant growth and yield. For example, multi-walled carbon nanoparticles (MWCNP) have been shown to promote seed germination and growth of tomato and enhance the growth of tobacco cells. The same phenomenon was observed in MWCNTs in mustard plants. Using the so-called germination index and relative time of root elongation as etalon parameters, it was shown that oxidized MWCNPs exert a better effect at lower concentration than non-oxidized ones.

Comparative studies for evaluation of the seed yield and prevention of leaf abscission in borage plant treated with nanosilver and silver nitrate have shown that the former performs better. It is known that the plant hormone ethylene plays a key role in leaf abscission, and silver ions inhibit ethylene by replacing copper ions from the receptors. When the both compounds were applied on the plants through the foliar spray method, it was observed that nanosilver was effective at a lower concentration than silver nitrate. Similar promoting effects of biosynthesized silver nanoparticles on the emergence of seedlings and various plant growth parameters of many economically important plant species have been reported.

Various studies have been performed to clarify the effect of ZnO nanoparticles on the growth of different plants. Thus, a stimulatory effect has been shown on the growth of Vigna radiata and Cicer arietinum. ZnO nanoparticles adsorption on the root surface was observed through correlative light and scanning electron microscopy and adsorption by the seedlings, by inductively coupled plasma/atomic emission spectroscopy. The effect of ZnO nanoparticles on plant cell physiology was investigated using the cellular antioxidant system as a model. Applying the foliar spray method on chickpea seedlings, it was shown that low concentrations of ZnO nanoparticles have a positive effect on plant growth and that the seedlings biomass accumulation has improved, which may be due to lower reactive oxygen species (ROS) levels (evidenced by the lower malondialdehyde content). Field experiments confirmed that application of ZnO nanoparticles at a dose 15-fold lower than the recommended dose of ZnSO₄ led to 29.5 % higher pod yield.

Comparable positive effects of ZnO and CeO₂ nanoparticles on Cucumis sativus fruit quality have been observed. The application of both nanoparticles resulted in increased starch content and possibly, in an altered carbohydrate pattern.

Stimulation of the antioxidant activity and nitrate reductase by a mixture of SiO₂ and TiO₂ nanoparticles in G. max has been found, in addition to the better productive effect and increase in water and fertilizer uptake capacity of the model plant. The application of TiO₂ nanoparticles has been demonstrated to promote photosynthesis under both visible and ultraviolet light and growth in spinach. An increase of 73 % in dry weight, three-fold higher photosynthetic rate, and 45 % increment in chlorophyll after seed treatment in spinach were observed. The authors speculate that the increment in the photosynthetic rate may be due to the increase in the absorption of inorganic nutrients, which enhances the utilization of organic substances and the quenching of oxygen-free radicals.

Unlike most nanoparticles, whose application at high concentration is not recommended due to the observed negative impact, TiO₂ nanoparticles applied at concentrations as high as 2,000 ppm increased the seed germination and seedling vigour in Brassica napus. Hence, different metal nanoparticles obviously show positive influence at various concentration ranges, e.g. Pd and Au at lower concentration, Si and Cu at higher concentration, and Au and Cu in combined mixture. This behavioural patter has been confirmed in field studies with G. max and Brassica juncea: nanocrystalline powder of iron, cobalt, and copper at an extra-low concentration promoted the seed germination rate, and a marked increase in the chlorophyll index, number of nodules, and crop yield was observed. Similarly, foliar spray of gold on plants in field experiments showed positive effects resulting in increased plant height, stem diameter, number of branches, number of pods and seed yield, and, interestingly, improved the redox status of the treated plants.

Ethical and safety issues of nano-fertilizers application

Undoubtedly nanotechnology has incredible potential to revolutionize many aspects of human life. However, the advancement of this multidisciplinary branch of science, especially the benefits from the practical application of nanoparticles have to be considered with some precautions.

The major concern at world scale is whether the unknown risks of nanoparticles involving their environmental and health impact prevail over their potential benefits. Thus, the risks associated with the application of nanoparticles are yet to be evaluated before the application of nanoparticles is fully accepted and implemented. Hence, “nanotoxicology” has been developed, which is responsible for assessment of the toxicological potential and promoting safe design and use of nanoparticles. Due to the thorough quantitative analysis of the potential health impacts, environmental clearance, and safe disposal of nanoparticles, improvements in the design of further applications of nanotechnology can be anticipated.

No direct human disease has been linked to nanoparticles so far. Nanoparticles, which constitute a part of ultrafine particulate matter, can enter in the body of humans/animal through the oral, respiratory or intradermal route. Currently, there is a common assumption that the small size of nanoparticles allows them to easily enter tissues, cells, and organelles and interact with functional biomolecular structures (i.e. DNA, ribosomes), since the actual physical size of an engineered nanostructure is similar to many biological molecules (e.g. antibodies and proteins) and structures (e.g. viruses).

Of course, there is still a need for proper physicochemical characterization and determination of appropriate exposure protocols and reliable methods for assessing nanoparticles outcome in the environment, their internalization, and their kinetics in living organisms. These are the prerequisites for establishment of optimal experimental conditions that will allow precise determination if a particular nanoparticle poses a threat to human health. However, the interdisciplinary research of materials scientists, environmentalists, and life scientists is contributing to the identification of the true, if any, hazards of nanotechnology. The heterogeneous and developmental nature of nanotechnology is making risk assessment quite subjective. The absence of standardized methodologies and guidelines makes it difficult to compare the safety/toxicity assessments from different research groups. It is most likely that different types of nanoparticles vary as to their toxicological properties. To interpret correctly any toxicological data, it is essential to calculate and determine the expected concentrations of nanoparticles that may be exposed to the biological system or present in the ecosystem. The risk assessment of nanoparticles has to be performed on a case-by case basis. Thus, the ethical issues must be specific for a specified product at a given time, and alternative assessments are needed to take into consideration ethical, social, and political values that relate policies such as those involving nanotechnology.

The use of nanotechnology in agriculture is very important, as it directly affects humans. Nano-fertilizers enable nanoparticles to enter the food chain, allowing their distribution in every organism related to the food chain. Literally all substances can be toxic to plants, animals or humans at some exposure level. However, this does not limit their use in various applications which are formulated minding the critical exposure concentration. As mentioned above, the promoting effect of nanoparticles on plant growth and physiology is expressed at very low concentrations; hence, it is hard to believe that these concentrations will pose significant health and environmental damage.

Many countries have identified the potential of nanotechnology in the food and agriculture sectors. Meanwhile, they recognize the need for assessment of the food safety implications of nanotechnology. As suggested by the scientific committee of the European Food Security Authority (EFSA), “the risk assessment paradigm (hazard identification, hazard characterization, exposure assessment and risk characterization) is applicable for nanoparticles (EFSA Scientific Committee 2011). However, risk assessment of these nanoparticles in the food and feed area should consider the specific properties of the subject nanoparticles in addition to those common to the equivalent non-nanoforms.”

Deciding the risk associated with the use of particular nanoparticles in food and feed means taking into consideration various parameters, among which physicochemical characterization of nanoparticles, their stability in the food and feed, toxicokinetics (absorption, distribution, metabolism/biotransformation, excretion/elimination) within the human and animal systems.

Genetically engineered microbes as biofertilizers

Genetically modified bacteria for agricultural purposes

There are numerous bacterial genera whose representatives can influence plant growth and production. Among these representatives, there are pathogens that can suppress plant diseases and they are used as biocontrol strains. Another group or bacterial species can contribute to increased plant growth by enhancing the availability of nutrients. These bacteria constitute the bio-fertilizers and are known as growth-promoting rhizobacteria (PGPR) as well. The term PGPR is associated with the ability of these bacteria to grow well at the interface between soil and plant root (the rhizosphere). PGPR can be applied either as seed coating or directly to soil. However, to exert their growth-promoting effect, sufficient numbers of the introduced PGPR have to survive in soil and rhizosphere, which not always happens. Consequently, the efficacy of PGPR is not always sufficient for commercial applications and there is a need to improve their performance. One of the possible approaches is to apply genetic modifications to facilitate the survival efficiency.

Survival of genetically modified bacteria in soil

Any microbial cell introduced into the environment will encounter a large number of biotic and abiotic factors affecting its survival. Both biotic and abiotic factors are equally important. Thus, high clay content, high pH, and relatively high moisture content can have a positive effect on bacterial survival. On the contrary, dry periods, presence of competing microorganisms, predation by protozoa, and lysis by bacteriophages negatively affect the number of introduced bacteria. Speaking of biotic factors affecting the activity and survival of introduced bacteria, the presence of plant roots that provide nutrients to the microorganisms living in their vicinity is very important. Among the microorganisms that are well adapted to the rhizosphere, there are members of the genera Agrobacterium, Azospirillum, Azotobacter, Bacillus, Erwinia, Pseudomonas, Rhizobium, and Xanthomonas.

Microbial survival depends on the interrelation between the environmental conditions and the physiological state of the bacteria. As a result of these interactions, bacterial cells can switch their metabolism to different physiological states. For instance, cells can become more stress resistant or form dwarf cells, they can produce exopolysaccharides for protection, they can enter a viable but non-culturable state, and some are able to form spores or associations with plants.

One can speculate that the survival pattern of the GM bacteria will follow the one of their wild-type parents. In fact, this extrapolation should be applied with some precautions. Firstly, the expression of the inserted genes requires an extra amount of energy, which could reduce their environmental fitness. In addition, the insertion could have disrupted unknown functions weakening the competitiveness of the strains. Secondly, it is possible for the GMMs to evolve and adapt to the prevailing environmental conditions via natural selection. This last statement is supported by evidence for evolutionary adaptation of bacteria to degrade the herbicide 2,4-dichlorophenoxyacetic acid resulting in increased competitive fitness to use succinate as a substrate. Similarly, it has been reported that environmental stresses could alleviate the debilitating effects of mutations: organisms may become more tolerant to genetic perturbations under certain environmental stresses.

GMMs have been shown to survive even better than the wild-type strain in studies under artificial growth conditions. However, enhanced survival of GMMs has rarely been observed under field conditions. Often, the population of introduced bacterial cells declines rapidly in soil, and the GMM species survive in a mode similar to that of non-modified bacteria. There are a lot of experimental studies in which no difference in survival between GMM and the parent strain could be detected (for Pseudomonas chlororaphis, P. fiuorescens and Sinorhizobium meliloti). Furthermore, some GMMs were reported to be out­competed by the parent strains. It is speculated that the presence of a number of constitutively expressed marker genes in a GMM had a negative effect on its survival in competition with the wild­type strain. Most probably, it is the metabolic load that is responsible for the decreased fitness, since this effect does not occur under nutrient-rich conditions.

To correctly interpret bacterial survival data, it is of crucial importance to use a reliable method for detection, since cells that enter a non-culturable state cannot be detected with standard cultivation-based techniques. Moreover, various studies have shown that GMMs introduced into soil become non-culturable. The presence of viable but non-culturable cells, dead cells or naked DNA detected using molecular techniques contributes to the complexity and the ecological significance of GMMs and their fitness in the context of the effect of the genetic modification introduced. The reliable way in which the effect of small differences in fitness will be measurable is to co-inoculate a GMM and its parental strain placing them in direct competition. However, results from such direct competition experiments have to be interpreted with care as well, since commercial application of GMMs does not include direct competition between GMMs and their wild-type strains.

All these data, contradictory to some extent, show that a conclusion regarding the survival of GMMs as compared to their parental strains cannot be definitely drawn. In each case where the colonizing ability and survival of the GMM are of importance, these parameters will have to be determined.

Environmental impact of GMMs inoculated into soil

The possible effects of the release of GMMs in natural microbial ecosystems are quite diverse. The range encompasses events such as input of organic substrate, displacement of species, changes in population structure, and possible loss of certain functions; production of toxic metabolites, which might lead to disturbance of key ecological processes. It should be taken into consideration that small changes in community composition are difficult or even impossible to determine, and the relationship between microbial diversity and ecosystem functioning is not quite clear. Undoubtedly, the soil microbial diversity is enormous, with a high redundancy of functions. Disappearance of a few species with certain functions will be difficult to detect, since many functions can be performed by a large number of different microbes. Thus, only extreme disturbances might affect soil microbial communities to the extent that certain functions will be negatively influenced.

The limited culturability of indigenous soil microflora is one of the major problems in microbial ecology. DNA- and RNA-based techniques, which do not involve cultivation of microorganisms, are currently used to detect the impact of GMMs on the indigenous microbial community. The methods that are suitable to analyze shifts in community structures include denaturing gradient gel electrophoresis (DGGE), amplified ribosomal DNA restriction analysis (ARDRA), terminal restriction fragment length polymorphisms (T-RFLP), and single-strand conformation polymorphism (SSCP).

Fate and effect of bio-fertilizer strains – field release

GM derivatives of bacteria contribute to an enhanced nutrient availability for plants, and thereby increase plant growth.

The most important bio-fertilizers are bacteria, such as Azospirillum and Rhizobium that can fix nitrogen. Rhizobium, Bradyrhizobium, and Sinorhizobium are plant symbionts, which form root nodules in leguminous plants and fix atmospheric nitrogen. These bacteria have been used widely as plant inoculants to increase the yield of leguminous crops. There is a long history of safe use of non-modified rhizobia as inoculants to increase crop yields. However, the yield increase is variable, and the success of inoculants seems to be dependent on competition with indigenous strains that are usually less effective. Rhizobium, Bradyrhizobium, and Sinorhizobium have been reported to survive in soil for years, in some cases even without the presence of their specific host. Rhizobium has been shown to be able to form nodules when its host plant is planted again after several years. This shows that presence of the host plant is not strictly necessary for their survival, but also characteristics of the strain not related to symbiosis play a role in its survival in bulk soil for years. Fast-growing Rhizobium species have been found to be more susceptible to desiccation than the slower-growing Bradyrhizobium.

Genetically modified Azospirillum and Rhizobium strains

Except for carbon dioxide (CO₂) which plants obtain from the atmosphere, plants get all their nutrients from soil. Nature has developed various mechanisms to supply plant nutrients by means of renewable resources, and the best example of this principle is biological nitrogen fixation in leguminous plants. Nitrogen-fixing bacteria can be regarded as a self-propagating source of nitrogen for plants. Unfortunately, not all plants are able to perform such interaction with N_2-fixing bacteria. That is why, at present, plant production yields still largely depend on input of chemical fertilizers. Most of these fertilizers are very mobile in the soil and are supplied in greater quantities than required for optimal plant growth. The loss of valuable compounds is not only of economic importance; this also causes serious problems for the environment, through leakage in surface and ground water and accumulation in the atmosphere.

Different strategies have been developed that aim at better uptake of fertilizers by plant roots. These include other formulations of fertilizer (e.g. slow-release fertilizer) and the use of plant-growth-promoting rhizobacteria (PGPR).

PGPR can exert their effect in both direct and indirect way. The indirect pattern comprises exercise of biocontrol of pathogens and deleterious microorganisms. The best documented example of PGPR acting in a direct plant-growth-promoting way is phytostimulation. Various bacterial genera are capable of producing plant-growth-stimulating factors (auxins, cytokinins, etc.) and when colonizing the roots of plants, they promote root growth. This assures a better uptake of water and nutrients by the plants and can result in higher crop yields.

GM Azospirillum increases nitrogen uptake

It is known that Azospirillum strains can promote plant root development and increase nitrogen uptake through the phytohormones produced by them. However, the mechanisms by which, and the conditions under which, these bacteria produce phytohormones as well as the interaction between bacteria and plant roots, are still not defined and require a better understanding.

To elucidate these mechanisms, several important questions/approaches should be addressed:

• The genetic and biochemical grounds of the synthesis of indole-3-acetic acid (IAA), the plant-growth-promoting hormone produced by Azospirillum;
• The construction of genetically modified Azospirillum stains with known production levels of IAA (i.e. IAA-minus , IAA-attenuated, IAA-over producers);
• Testing the effect of these genetically modified bacteria on plants (growth promotion, nitrogen uptake) and on the environment (interaction with resident microbial flora, survival and spread) under field conditions.

At present, GM Azospirillum strains with these basic features are available. Research with these strains is focused on their impact on resident microbial populations, plant growth and nitrogen uptake rates from soil. These studies are being conducted in lab experiments (i.e. growth cabinet and glasshouse studies) in order to gain vital information on the way GM strains are likely to behave under field conditions. The experiments are conducted with a range of crops, soil types and climate conditions, representing the existing agricultural parameters within Europe. Despite of the advancement of these research studies, extensive and careful testing under containment is required before the GM Azospirillum can be considered for field release.

GM Rhizobium strains with increased competitiveness

Legume inoculation with highly efficient nitrogen-fixing bacteria is a widely used approach to increase the productivity of leguminous crops. This inoculation is not always successful, since native soil bacteria with low nitrogen-fixing efficiency can out-compete the introduced strains in terms of nodulation initiation. What is critical for the successful use of rhizobial inoculants is their competitiveness, i.e. the ability to dominate nodulation. Thus, inoculant strains are modified in a way that they occupy a sufficient number of root nodules to provide high rates of nitrogen fixation for the plant host.

Experiments with Sinorhizobium meliloti strains from diverse geographical origins regarding their competitiveness for alfalfa roots have shown that, in all cases, this property has been enhanced by genetic manipulation. The said genetic manipulation comprises modification of the expression of the nifA gene which is responsible for the control of all the rest nitrogen-fixation (nif) genes. When GM S. meliloti strains were mixed with wild-type ones, the former occupied most of the nodules on the alfalfa roots. The precise mechanism of this improvement is not understood yet but it is speculated that nifA regulates the expression of genes different from the nif cluster, resulting in an advantage during nodule formation and development.

Another feature that contributes to the nodulation competitiveness of Rhizobium strains is their ability to efficiently recognize the plant root. This is very important because the efficient inoculation means lower doses of the bacterial strain. Furthermore, the movement of the inoculation strain towards the plant roots is another factor influencing competitiveness. Experiments with GM Rhizobium leguminosarum strains engineered to express the β-glucuronidase reporter gene (gusA) showed that the percentage of the nodules induced by the GM gusA­labeled strain is higher compared to the nodules induced by a flagella-deficient non-motile strain. In this way it was proven that the functional flagella are required for effective competition for nodulation.

All these data provide valuable information on the mechanism of root attraction allowing the development of Rhizobium strains with enhanced nodulation competitiveness and increased host specificity.

Impact of GM Rhizobium strains on arbuscular mycorrhizal fungi

Arbuscular micorrhizal fungi are an important group of fungi that form symbiotic relationships with plants. A major question is whether the application of GM Rhizobium strains with increased competitiveness leads to an increase in the colonization and nodulation of the plant root or it interferes with the beneficial symbiotic relationship.

In lab and green-house experiments, it has been shown that a GM Sinorhizobium meliloti strain with improved nodulation ability does not interfere with any aspect of mycorrhizal formation by the representative AM fungus Glomus mosseae. On the contrary, GM S. meliloti increased the number of AM colonization units and the nutrient acquisition ability of the mycorrhizal plant.

GM Rhizobium strains: field release

Several Rhizobium species have been genetically modified either to improve nitrogen fixation, or to study their survival, making use of marker genes through field trials.

Thus, a Tn5-marked R. leguminosarum strain introduced into a field as an inoculant for peas and cereals persisted for 5 years in the plots where peas were grown. The persistence of the strain was attributed to the soil type, the cultivation of the proper host plants, and the climate conditions. Potential non-target effects on the microbial ecosystem were not studied.

The use of an improved R. meliloti strain, with additional copies of nifA and dctABC, resulted in a 12.9% increase in alfalfa yield in a field study. However, at sites with high nitrogen concentrations or native rhizobial populations, the alfalfa yield did not increase.

The fate of a Tn903-marked R. meliloti strain introduced into alfalfa-planted field plots was studied and it was found that the cell numbers decreased rapidly after inoculation. One year after introduction, the numbers of introduced cells had dropped to below the numbers of indigenous rhizobia.

In a contained field experiment, a GM S. meliloti strain with enhanced competitiveness for nodule occupancy was released in the rhizosphere of alfalfa. Effects of the GMM and the wild type on the indigenous microbial communities were studied by restriction fragment length polymorphism (RFLP) and temperature gradient gel electrophoresis (TGGE). Inoculation of wild type and GMM had only limited effects. It appeared that alfalfa plants had a greater influence on the microbial community than the inoculated strains.

Both the fate and ecosystem effects of a Luc-marked S. meliloti in a field experiment with Medicago sativa were studied. The bacteria were detected up to 12 weeks after introduction. No effects of the strains on carbon and nitrogen concentrations in the soil could be detected, and there were no differences in the total number of colony-forming units of indigenous microorganisms. Over a thousand bacterial isolates obtained from the plots were further studied by ARDRA, and the dominant groups were identified by 16S rRNA sequencing. In the rhizosphere of M. sativa, the numbers of Alcaligenes and Pseudomonas were reduced as a result of the inoculation. Molecular analysis by studying the SSCP banding profiles revealed shifts confirming the effect of the inoculum on the native microbial population.

In China, wild-type and GM Alcaligenes faecalis isolates have been introduced into rice fields at a large scale to improve crop productivity. A. faecalis, a non-nodule-forming nitrogen-fixing isolate, was genetically modified by insertion of a constitutively expressed nifA regulatory gene. Nitrogen fixation appeared to be 15–20% higher and the yield was 5–12% higher compared to the non-treated fields. The possible ecosystem effects of the introduction of this GM strain was studied by DGGE of amplified 16S rDNA in a microcosm experiment. The introduced GM strain survived well in the rhizosphere. The DGGE banding profiles of samples treated with the modified strain closely resembled the profiles of untreated samples throughout the 40 days of the experiment, suggesting that there are no obvious effects on the bacterial community. Overall, the survival of the strain and the increase in crop yield indicate that this derivative of A. faecalis is a good candidate for commercial application, since its ecosystem effects seem very limited.

The impact and fate under field conditions of GM Rhizobium strains were investigated in a field trial with a model system comprising different GM Rhizobium leguminosarum v. viciae strains, marked with the lacZ gene and HgCb resistance genes (mer genes) inoculated in the rhizosphere of pea plants. Three modified strains were used:

• 1110 strain containing plasmid pDG3 carrying genes for resistance to HgCb (mer genes) and lacZ whose expression is under the control of the lacZ-lacO system;
• 1111 strain carrying the plasmid pDG4 in which the lacZ gene is constitutively expressed at high levels;
• 1112 strain containing a copy of mer genes and a regulated lacZ gene inserted into the chromosome.
• Wild-type R. leguminosarum v. viciae 1003 was used as a control.

These strains were monitored according to the reporter lacZ/mer system along with the soil metabolic activity plus nitrogen-transforming capacity.

The field experiments showed that all tested strains colonized the rhizosphere to the same extent; similar values were determined for the respiration rate and soil metabolic activity as well as for the nitrogen-transforming capacity of all tested strains. These results indicate that, although the presence of the plant had a considerable impact on carbon mineralization in soil, the impact of GM Rhizobium strains is indistinguishable from the impact of the wild-type strain, and also suggest that the impact of the plant on microbial activity is considerably greater than the impact of GM inoculants compared with wild-type strains.

In spite of the fact that the field trials with GM bio-fertilizers are limited, the initial results about their use are promising with respect to the improved performance in agricultural applications. GM bio-fertilizers have been introduced with an encouraging success in terms of the survival and activity of the inoculants, which is dependent on the environmental conditions. So far, non-target effects of GM bio-fertilizer strains that have been reported are small and insignificant compared to natural variations, such as differences between populations of different plant species.

However, our knowledge on the benefits, fate and effects of GM strains in the environment is still quite limited and partial.

Questions that have to be solved include: how and when (at what physiological state) bacteria survive best in soil; what their effect on the natural microflora is; how a mixed microbial community can be structured and optimized for use in agriculture. And last but not least, what the ecosystem effects of GM strains are, especially on non-target organisms.

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