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Changes in phytoremediation are observed mostly in terms of plant features. Hyperaccumulators able to accumulate above average amounts of pollutants are of prime concern in the area (Memon and Schröder 2009). As the next step, a combination of phytoaccumulation with high and fast efficiency in plant biomass production is important to improve the process rationalization (simpler harvest of plants), and to increase the accumulation with the perspective of wood utilization in energy production. Results presented recently underline physiological and biochemical aspects of phytoremediation with the influence of environmental factors (plant-fungus or plant-soil interactions) with possibilities of more efficient implementation of this process. An interesting prospect in this scope may be the exploration of combined techniques (e.g. selected plants with phytoremediation abilities with such technical or semi-technical methods as electrokinetic remediation under constant voltage across the soil).
Heavy metals belong to an ecologically significant and toxicologically unique class of toxicants, because they are spread everywhere, particularly in industrialized areas. Effective phytoextraction requires the regular (not significantly inhibited) growth of plants in polluted areas followed by the activation of defence mechanisms. Plants with phytoremediation abilities have to meet several fundamental criteria: high effectiveness of phytoaccumulation/phytodegradation, high biomass increase, well developed root system, high resistance to pollutants, easy adaptation to different environmental conditions and simple environmental requirements (Vangronsveld et al. 2009). Plants used in phytoremediation should exhibit no or very small risk of metals' transport to higher trophic levels (reduced possibility to contaminate the food chain).
To date the following topics have been elucidated and are well known: removal of pollutants by aerial plant organs, transport of metals through the plasmalemma, and detoxification of pollutants in the cell (chaperones, phytochelatins, metallothioneins, low molecular weight organic acids - LMWOAs) (Pal and Rai 2010). The above possibilities allow plants to defend themselves against viruses, microbes and fungi with effective phytoaccumulation/phytodegradation of pollutants present in the environment (Rascio and Navari-Izzo 2011).
6 Ecological (mycorrhizal) and chemical (Ca/Mg ratio, LMWOAs) factors influencing phytoremediation efficiency
Phytoremediation efficiency depends on a complex sequence of factors influencing the number of interactions, one of the most important being the species/variety of used plant. In different studies significant diversity in the phytoextraction/phytodegradation effectiveness was confirmed, not only within the species but also among varieties of the same plant species (Mleczek et al. 2010). The most important differences of plant traits are as follows: structure, size, immunity and individual traits such as environmental and climatic requirements (water, nutrients or temperature).
Natural bioremediation with selected bacterial strains and fungi is an interesting solution in decontamination of areas polluted with organic compounds (Juwarkar, Singh and Mudhoo 2010). Like other methods, bioremediation is also limited (by interaction of microbes with existing microorganisms, presence of toxic substances inhibiting microbial development or low bioavailability of xenobiotics). Hence cooperation of both methods has a significant role in increase of phytoremediation efficiency. Along with many strategies focused on plants' accommodation to unfriendly pollutants, a symbiosis with mycorrhizal fungi seems to be very helpful (Vamerali, Bandiera and Mosca 2010). Fungi in the rhizosphere are a significant factor in phytoaccumulation/phytodegradation efficiency increase, to stimulate plant growth as well as to increase the resistance of plants to concentrations of pollutants found in the environment. The synergism is especially significant in the case of hyperaccumulators. According to the literature data mentioned above, more than 400 plant species are documented as hyperaccumulators and they belong to the following families: Asteraceae, Brassicaceae, Caryophyllaceae, Cyperaceae, Cununiaceae, Fabaceae, Flacourtiaceae, Lamiaceae, Poaceae, Violaceae and Euphorbiaceae.
The presence of microbial clusters (different genes in rhizoremediation) may decrease levels of plant stress hormone. Especially significant is a combination of plant and plant growth promoting rhizobacteria (PGPR). Inoculation of selected plant species (including hyperaccumulators) with endophytic bacteria, e.g. Achromobacter xylosoxidans, Bacillus pumilus, Corynebacterium flavescens, protects plants against the phytotoxic effects of heavy metals and/or different xenobiotics (Glick 2010). In this case a significant role of plants able to exude pollutant-degrading enzymes into the rhizosphere can be found in plants, fungi, endophytic bacteria and root-colonizing bacteria (e.g. root-specific laccase (LAC1), peroxidases, haloalkane dehydrogenase (DhaA), P450 monooxygenases, phosphatases, nitrilases). These enzymes are able to transform pollutants without their uptake (Dowling and Doty 2009; Gerhardt et al. 2009).
The efficiency of selected heavy metals' phytoaccumulation depends on the mutual relations of macroelements important in plant growth (nutrition) and development in the polluted matrix. When macroelements are present in excess or deficiency, oxidative stress begins but also different ratios of them are important in the phytoremediation process. As an example, when compared to the physiological Ca/Mg ratio (4:1), an increase of calcium ion concentration in relation to magnesium ions in studied soil (Mleczek et al. 2011) resulted in decreased cadmium and lead phytoextraction efficiency by Salix viminalis 'Cinamomea'. Additionally, Salix growth was restrained under 1:10 Ca/Mg ratio while it was stimulated under 20:1 ratio, which is opposite to cadmium and lead sorption.
Additionally, a change of Ca/Mg ratio influences the amount and kind of low molecular weight organic acids (LMWOAs) exuded into the rhizosphere. A model experiment where the efficiency of formation of selected LMWOAs depending on cadmium, copper, lead and zinc concentration was tested and indicated selective exudation of acids depending on the concentration and the kind of heavy metal. In physiological 4:1 Ca/Mg ratio the following acids formed complexes with particular heavy metal ions: citric, lactic, maleic and succinic acids with Zn2+, and malonic acid with Pb2+ and Zn2+. A change of Ca/Mg ratio to 1:10 caused that citric (Cd2+, Zn2+ complexation), maleic and succinic (Cd2+, Cu2+, Pb2+, Zn2+) acids were observed in the rhizosphere (Magdziak et al. 2011).
The rhizosphere, as the space in the immediate vicinity of roots, is permanently influenced by their exudates. Moreover, it differs - in relation to other soil fractions - in the composition and large amounts of bacterial cells (the phenomenon of bacteriolysis) and fungi (mycorrhiza), with diversified decay of plant roots, soil structure, composition of organic matter, pH, humidity and microorganism activity. The properties often change in a particular site, demonstrating frequently dynamic changes in time (Macek et al. 2007). All the above-listed factors influence the solubility and uptake of pollutants, both indirectly, through the change of their microbiological activity and the root growth dynamics, and also directly through changes of soil reactions, chelation, precipitation of deposits and oxidation-reduction reactions.
In the broad spectrum of organic compounds present in the rhizosphere, particular attention is focused now on LMWOAs. Organic acids such as malic, oxalic, acetic or citric are recognized as the most significant ones in many different processes in the rhizosphere. Depending on their degree of dissociation (efficiency), and the amount of carboxylic groups in the molecule, acids can appear in the form of differently charged anions, which in consequence results in the possibility of metal cations' complexation and relocation from the soil. This is the reason that acids are reported as components of the soil environment which in the rhizosphere take part in many processes, e.g. in dissolving and uptake of nutrients (e.g. P and Fe) by plants and microorganisms, decrease of stress associated with anaerobic conditions, dissolving soil minerals leading to pedogenesis, and detoxification of heavy metals by plants (e.g. Al).
LMWOAs exuded by plant roots play a significant role in bacterial microflora composition in relation to nutrients and amounts of available forms of elements in the soil (Magdziak et al. 2011). Moreover, acids influence decomposition of organic matter, and structural formation with particular physical-chemical soil properties. Heavy metals are present in polluted soil in a form insoluble in water, because afore-mentioned water-soluble LMWOAs, as rhizosphere components excluded by the plant root system, change the rhizosphere features, which results in heavy metals' complexation to insoluble forms in soil. LMWOAs usually appear as anions. This allows for instantaneous reaction with metal ions, in the water phase, soil solution and in constant phases, which makes it an important element in the phytoremediation process. It is worth underlining that interaction of organic acids with metals and other elements closely depends on the kind of soil. For example, for a nutrient such as phosphorus, dissolving and mobilization of ions of this element by selected LMWOAs (oxalic and citric acids) is closely related to the soil type. Similar relations exist for other exuded LMWOAs playing a significant role in macroelement mobility increase (e.g. Cu, Cd, Zn) and mechanisms of heavy metal immobilization (Al, Cd, Ni).
The problems presented above and associated with the type of soil and its physical-chemical properties indicate ambiguous information about LMWOAs' function in the rhizosphere. Some data inform about mobilization and elution from the soil of heavy metals after soluble complex formation with the acids, but this information is fragmentary and insufficient to answer the following questions: (i) do organic acids released by roots influence the mobilization and uptake of heavy metals by plants from the rhizosphere, or (ii) is their amount dependent on the concentration and chemical character of the metal, or (iii) does the amount of acids indicate activation of the plant defence mechanism against stress? Such information can also elucidate the role of plant genetic factors in increase of heavy metals' availability and uptake from soil followed by improved effectiveness of heavy metal accumulation. For that reason more detailed studies are needed to fully answer the above questions.
7 Conclusions and future perspectives in phytoremediation
To conclude, phytoremediation - being one of the most interesting methods of environment cleaning - seeks for an alternative solution to hyperaccumulators or high biomass green plants. Symbiosis between plants and bacteria or fungi together with genetically modified plants are of the greatest chance for phytoremediation development. The major concern of the process efficiency is plants' resistance to environmental stress factors represented by the pollutant its self (heavy metal, xenobiotic), as well as by accompanying stressors (air pollution at contaminated site, fungal infections, etc.). Thus, the understanding of oxidative stress and defence mechanisms of plants used in phytoremediation is of a great importance. In our studies, tropospheric ozone induced the biosynthesis of salicylic acid in ozone-sensitive tobacco plants which was strongly correlated with the level of injuries observed on leaves after exposure in ambient air conditions. We could assume, that ozone has a strong negative impact on plants causing the ozone-induced oxidative burst.
Several secondary metabolites are synthesized by fungi during morphological and metabolic transitions, when the accumulation of ROS occurs (e.g. Aspergillus parasiticus) what effects in oxidative stress and mycotoxins (e.g. aflatoxin) biosynthesis. In conclusion, we believe that oxidative stress promotes secondary metabolism and mycotoxins (secondary metabolites) are part of differentiation process in fungi. On the other hand, plant compounds involved in plant-fungi interactions are able to interfere with mycotoxin biosynthesis in host tissues.
An ideal plant with all traits important in phytoremediation is not available in the environment, which indicates the need for the introduction of new genetically modified organisms (GMO) and their application in phytoremediation. It is believed that through modification of the plant genome, by implanting DNA of one plant in another organism, and thus obtaining a new plant with significantly improved phytoremediation abilities, it will be possible to introduce into practice more valuable plant material. To construct the perfect hyperaccumulator, it is necessary to elucidate the complex of mechanisms in the field, to meet all the basics of phytoremediation. Depending on the goals, selection of important genes followed by model experiments (hydroponic, aeroponic) should be the first step in studies on phytoextraction/phytodegradation efficiency.
The literature in recent years indicates that genes coding heavy metal ion transporters and associated ligands are of prime concern. Generally, it is possible to underline a few fundamental types of genetic plant modifications including increase of resistance to herbicides (two enzyme systems have a significant role: cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs)), insects, diseases caused by fungi and viruses, as well as unfavourable environmental conditions (Dowling and Doty 2009). Probably all these modifications will be used to improve plants' phytoremediation abilities required depending on the site and conditions of the process (Kawahigashi 2009).
A significant influence on the heavy metal phytoaccumulation efficiency and plant resistance in the case of high levels of heavy metals and other pollutants in the environment is exerted by protein origin heavy metal chelators such as phytochelatins and metallothioneins (MT). Gene modifications responsible for phytochelatins or glutathione (GSH, c-L-glutamyl-L-cysteinylglycine) synthesis next to metallothionein genes will probably exemplify one of the most important ways of preparing transgenic plants in the near future (Dowling and Doty 2009). Transport of heavy metal ions from the root system to shoots or leaves requires the presence of specific transport proteins. Additionally, some enzymes essential in biotransformation are able to catalyze the oxidation of toxic heavy metal ions (e.g. As3+, Cr6+, Sb3+), as shown by the new results of various studies (published recently worldwide) indicating new heavy metal ion transporters.
So far, several transporter families have been studied: ABC (ATP-Binding Cassette) - YCF1, ATPases type P (P1B) - AtHMA4, CDF (Cation Diffusion Facilitator) - cdf1, cdf2, Nramp (Natural resistance associated macrophage protein), YSL (Yellow Stripe Like) - YS1, ZIP (Zinc-regulated transporter (ZRT), Iron-regulated transporter (IRT)- like Protein) - AtZIP1 (Memon and Schröder 2009). The transporters are selective to pollutants but on the other hand being located in the cell membrane, or in membranes of the endoplasmic reticulum and vacuole, also transport essential elements, necessary in normal growth and development of the plant (Ca2+, K+, Mg2+, N, P and S), or microelements (Cu2+, Fe2+, Mn2+, Mo2+, Ni2+, Zn2+) including in this group toxic heavy metal ions (Cd2+, Co2+, Cr3+, Cr6+ or Pb2+).
An interesting and ambitious challenge in the area of genetic engineering for phytoremediation is the creation of systems consisting of transgenic plants with bacterial genes. Single-celled prokaryote microorganisms have a significant potential in genes responsible for mechanisms of heavy metal detoxification. Additionally, studies on the role and influence of autochthonic bacteria and mycorrhizal fungus in phytoremediation efficiency will help to enrich and improve the knowledge concerning mechanisms and basics of natural remediation (Yadav et al. 2010).
Probably controversies concerning genetically modified organisms in the case of GM plants applied in phytoremediation will not take place. Since plant hyperaccumulators usually are not in the food chain, it is believed that because of the lack of direct contact, proteins formed during transgene expression will not modify cell metabolism and cause harmful effects to human beings. It is also worth pointing out the dynamic increase of genetic engineering known in farm products (transgenic rice (Oryza sativa) - human cytochrome 450 gene CYP1A1 or human genes encoding human CYP1A1, CYP2B6, and/or CYP2C19 in rice (Abhilash, Jamil and Singh 2009) through agrobacterium-mediated transformation or transgenic tobacco - Enterobacter cloacae as a gene source with pentaerythritol tetranitrate reductase enzyme) introduced in phytoremediation studies (Brassica juncea, selected taxa of Populus, Arabidopsis thaliana or Phragmites australis) (James and Strand 2009). Another example is that of transgenic plants in phytodegradation of explosives (Jabeen, Ahmad and Iqbal 2009) by expression of bacterial nitroreductases and cytochrome P450s (e.g. glycerol trinitrate hexahydro-1,3,5-trinitro-1,3,5-triazine and 2,4,6-trinitrotoluene) (Eapen, Singh and D'Souza 2007). The above genes, especially in hyperaccumulators (Thlaspi, Brassica) in combination with significant (high) biomass production, will probably be very interesting trends in phytoremediation studies. The future in the field of genetic engineering in phytoremediation will undoubtedly necessitate a complex approach to this issue, but not only introduction of single genes/traits. In the case of plant-fungi or plant-microbe interactions, attention should be focused on mutual relations between microorganisms and rhizosphere components, including plant exudates. Playing a significant role in dynamics of phytoremediation development will be cooperation between many disciplines (novel gene and enzyme identification with metagenomics and genomic sequencing projects).
Considering the development of phytoremediation, the problem of polluted biomass should be considered. Plants in polluted areas usually exhibit concentrated pollutants, when compared with the relatively low level of pollutants in the soil. In the case of plants characterized by a significant biomass increase in combination with high phytoextraction efficiency, heavily polluted biomass is the final product. Such biomass is periodically collected; so the amount of it is significant and requires particular treatment before further utilization.
The amount of pollutants can be reduced by ocean dumping, deep well injection or approved secure landfills, but biomass volume can be reduced by physical, chemical, and thermal methods and with the use of selected microbial cultures. Phytoremediation as a green technology should not generate toxic substances and should improve the quality of the environment. Taking into consideration this important fact, it seems promising to apply thermo-chemical methods to utilize polluted biomass (pyrolysis, still works, combustion or gasification).
Considering that the phytoremediation process is characterized by significant efficiency of pollutants removed from the matrix and great biomass increase, this technology can generate additional financial benefits. The product of biomass thermal disintegration is ash, which has been an interesting subject of studies for many years with additional possibilities of phytoremediation application (phytomining - bioâ€‘ore). The biomass obtained after the phytoaccumulation process, including contaminants (heavy metals), can be the substrate in the steel industry, because there is a chance for their permanent recovery as valuable products (recovery of the heavy metals in pure form or in alloys).