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Novel biotechnological approaches in environmental remediation research
Biotechnology Advances 17 (1999) 679–687
Novel biotechnological approaches in environmental remediation research Marcia Pletsch*, Brancilene Santos de Araujo, Barry V. Charlwood Universidade Federal de Alagoas, CCEN, Departamento de Química, Campus Universitário, Tabuleiro dos Martins, 57072-970 Maceió, Alagoas, Brazil
Abstract Two novel approaches, the use of Agrobacterium-transformed plant roots and mycelia cultures of fungi, are considered as research tools in the study of the remediation of soil, groundwater, and biowastes. Transformed roots are excellent model systems for screening higher plants that are tolerant of various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants. Edible and/or medicinal fungi may also be natural environmental remediators. Liquid cultures of fungal mycelia are appropriate model systems with which to commence screening and biochemical studies in this under-researched area of biotransformation. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Phytoremediation; Bioremediation; Organic contaminants; Inorganic contaminants; Hairy roots; Mushrooms
1. Introduction Bioremediation and phytoremediation are terms used to describe those methodologies that employ living organisms to remove toxic compounds from the environment. While bioremediation refers to the use of lower organisms (bacteria and fungi) and phytoremediation refers to the use of plants, both technologies share the advantages of in situ application and environmental acceptability compared with most physicochemical strategies. Since the remediation approaches avoid having to transport the contaminated material to the site of treatment, large savings on the costs of handling, transportation, and storage of contaminated or waste materials may be achieved. Extensive reviews are available concerning the specific advantages, limitations and economics of bio- and phytoremediation [1–4]. The present article addresses two innovative as* Corresponding author. Tel: 155-82-2141386; fax: 155-82-2141615. E-mail address: [email protected] (M. Pletsch) 0734-9750/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0 7 3 4 - 9 7 5 0 ( 9 9 ) 0 0 0 2 8 -2
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pects of this subject: the application of Agrobacterium rhizogenes-transformed roots (hairy roots) in phytoremediation studies and the use of higher fungi (mushrooms) in bioremediation. Each one of these biological systems can contribute to our understanding of the mechanisms involved in the removal, containment, or decomposition of a wide variety of pollutants, particularly petroleum-based compounds, polychlorinated biphenols (PCBs), heavy metals, pesticide-related compounds, potentially toxic biowastes, and even radioactive contaminants. Despite the fact that transformed roots and mushroom mycelia are cytologically, morphologically, and biochemically diverse structures, in terms of remediation they carry out very similar functions. Both represent the hidden complex of a highly branched matrix of organized living cells, which is at the interface of the remediating organism and the contaminated substrate. Whatever is the fate of a contaminant in a plant or fungus or its surroundings, remediation always starts with uptake or release of active molecules by the roots and mycelia. 2. Phytoremediation and transformed roots Phytoremediation is being developed as an alternative technology for removing or, more accurately, reducing the concentration of toxic organic and inorganic pollutants in air, water, and soil. In recent years, an array of new terminology has emerged to designate the various processes involved in phytoremediation, such as phytoextraction, phytotransformation, phytovolatilization, phytostimulation, rhizofiltration, and phytostabilization, the principles of which are summarized in Table 1. Hairy roots have been obtained from numerous plant species following simple infection of sterile tissue with the soil-borne bacterium Agrobacterium rhizogenes. Most of the studies utilizing hairy roots have been concerned with the formation of secondary compounds of pharmaceutical value and during the past 15 years have provided a powerful tool for the elucidation of metabolic pathways, enzymatic steps, and regulation of secondary metabolism.
Table 1 Strategies in phytoremediation Type
Fate of contaminants
Reference
Phytoextraction
Absorption from the soil and accumulation into the roots (or other plant tissues) in an unchanged form for later harvesting (phytomining) Absorption and bioconversion (catabolism or anabolism) in the roots (or other plant tissues) Uptake and conversion into a volatile form that is released into the atmosphere Stimulation of microbial biodegradation by plant exudates Absorption from aqueous environments and subsequent disposal of root material Sequestration, lignification or humification in the soil
[28]
Phytotransformation Phytovolatilization Phytostimulation Rhizofiltration Phytostabilization
[23] [38] [39] [4] [2]
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However, the use of hairy roots is not only limited to the production of secondary products: the system can be applied to the improvement of rooting ability in recalcitrant species, production of useful plant proteins (commercially important enzymes, lectins, and ribosome inhibiting proteins), regeneration of transgenic plants exhibiting beneficial traits, and, more recently, phytoremediation. Hairy roots can contribute to research and development of methods for phytotransformation, phytostimulation, rhizofiltration, and even for phytoextraction where the contaminant is to be sequestered in root tissue. The specific advantages of using hairy roots as model systems are: (1) they may be obtained from a wide range of gymnosperms and angiosperms; (2) they grow fast in dilute, hormone-free media and in the absence of light and are hence rapid and cheap systems to implement; (3) they present genetic and biochemical stability similar to that of the mother plant; and (4) they are isolated systems that permit phytoremediation studies to be performed without the interference of the microbiota, or even of other plant organs. Regarding the host range of A. rhizogenes, it was thought for a number of years that within the angiosperms, only dicotyledonous plants were susceptible to Agrobacterium infection, while monocotyledonous plants were outside the host range of the bacterium. However, more recent advances in the understanding of the processes involved in the infection event (e.g. chemical signalling, organization of the virulence operon [5], physiological state of the explants, and their competence for transformation [6,7]) has led to both the construction of super-virulent strains of Agrobacterium [8] and more appropriate protocols for the transformation itself. Thus, many more monocotyledons now appear to be hosts for A. rhizogenes than previously thought, and this extends the boundaries for the application of transformed roots [9,10]. Even within the gymnosperms there are many successful examples of transformation with A. rhizogenes resulting in root formation and development (i.e. in various Pinus species [11,12]). A further possibility to increase the range of plants for which transformed roots may be obtained is provided by the demonstration by Kodama et al. [13] that hairy roots may be formed by the direct bombardment of plant material (in this case cotyledon tissues of Cucumis sativus) with a plasmid containing the rol genes of A. rhizogenes. The screening for high level accumulation of metals by hairy roots is a very practical operation because of the uncomplicated manner in which the experiments may be performed in the laboratory, under controlled conditions, thus eliminating the difficulties of conducting extensive trials in the field. That transformed roots retain the biochemical capacity of the parent plant is illustrated by the work of Doran and Nedelkoska [14], who showed that hairy roots of the well-known hyperaccumulating plants of the Cruciferae, namely Thlaspi caerulescens (which can contain over 3% zinc/cadmium) and species of Alyssum (which can accumulate up to 1% dry weight of nickel), retained their unusual ability to remove heavy metals from the culture medium. Extension of this type of search to discover other plants that might overaccumulate metals has led, for example, to the demonstration that hairy roots of horseradish (Armoracia rusticana) accumulated mercury, iron, and copper ions at high rates [15]. Apart from the elegant simplicity of using hairy roots for screening purposes, there is also the possibility of introducing new genes into roots through single-step transformations using genetically altered A. rhizogenes. The biochemical consequences promoted by the new gene product can then be analyzed directly in the transformed culture. Suitable target genes for such studies might be those encoding metal-binding proteins, such as metalloenzymes, met-
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allothioneins, metal storage proteins, and metal-activated enzymes, as well as those responsible for the synthesis of the phytochelatins, low molecular weight peptides with high affinities for metals [16]. Regarding organic contaminants, there is considerable evidence that demonstrates the feasibility of using transformed root cultures for the purpose of screening plants capable of degrading such pollutants and the subsequent identification of metabolic products. In one such example, Mackova et al. [17,18] showed that hairy root cultures of Armoracia rusticana, Solanum aviculare, Atropa belladona, and Solanum nigrum were able to degrade PCBs, with the last example presenting the highest metabolic capability, which lead to a reduction of PCB concentration in the medium from 100 to 40 ppm in 30 days. This efficient degradation of exogenous PCBs was associated with increased peroxidase activity in the root system. Furthermore, Hughes et al. [19] showed that the explosive 2,4,6-trinitrotoluene was converted by Catharanthus roseus hairy root cultures into minor products (i.e. aminated nitrotoluenes, and major, but uncharacterized, soluble products that were stored both within the root tissue and in the medium itself). Clearly it is very important to identify such catabolic products to determine the degree of toxicity in comparison with the original pollutant. Undoubtedly the hairy root system is very suitable for the determination of catabolic pathway reactions and their enzymatic regulation. Learning about the degradation pathways of specific organic pollutants in target plants is fundamental to the study of the overexpression of controlling genes and to the production of transgenic plants with enhanced degradative capabilities. Indeed, it is pointless to transfer a bacterial, fungal, or animal gene to a plant that would convert the contaminant into an intermediate that could not be stored or further transformed by the natural enzymatic machinery of that plant. When plants are subjected to physical or chemical stress or microbial or herbivore attack, their typical response involves the induction of either a new metabolic pathway or the augmentation of an existing pathway. Often, such elicited pathways lead to the formation of products (secondary metabolites) that form part of the plant defense mechanisms. Hairy roots have been a valuable tool in the study of the enzymatic regulation of such secondary pathways [20] and in the understanding of elicitor response mechanisms. Since pollutants constitute stressing agents to plants in general, the application of elicitation studies to hairy roots with the aim of determining which genes are triggered when tissues are exposed to specific contaminants will certainly be advantageous for phytoremediation technology. The early stages of xenobiotic exposure and its limiting factors (type and concentration of contaminant and length of exposure) must be investigated to understand how plants react in the face of chemical aggressors. We have investigated the effects of various concentrations of phenol, 2-chlorophenol, 2,6dichlorophenol, and 2,4,6-trichlorophenol on 30 clonal hairy root lines of Daucus carota [21]. The chlorinated derivatives were more toxic to the transformed carrot roots than phenol itself (i.e. root growth was possible in the presence of chlorophenols only at concentrations 20 times lower than that of phenol). The capacity of carrot roots to translocate phenols from the medium to the roots and further transform them was totally independent of the growth characteristics of the specific clones. This is an important point, since root clones may show distinctive biochemical attributes as reported for secondary compound accumulation. If hairy root cultures are to be used as model systems for screening tolerant plants capable of metab-
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olizing xenobiotics, then they must exhibit these properties independently of their origins (clonal or mixed cultures). Phenol and chlorinated phenols were able to elicit peroxidase activity by up to 100% in carrot hairy roots and this activity remained elevated, while the removal and metabolism of the major part of the phenols occurred [21]. It has also been shown [22] that pyrogallol, 2-aminophenol, p-cresol, and catechol elicit peroxidase activity in hairy root cultures of D. carota. Together with peroxidases, other enzymes of plant origin, such as dehalogenases, laccases, nitroreductases, and nitrilases, are reportedly involved in phytoremediation processes [23], some or all of which may be inducible by chemical stress. The situation concerning reductases, laccases, and dehalogenases in carrot roots is currently being investigated in our laboratory. Finally, hairy roots can be used to study root-microbe interactions and to understand how soil microorganisms are influenced by the presence of roots and/or their exudates. For example, the appropriateness of using plants to increase the activity of native or introduced microorganisms that can degrade xenobiotics (phytostimulation) and the conditions under which such a process may occur can readily be evaluated using the hairy root system. Experiments can be designed whereby roots may be inoculated with specific microorganisms, individually or in combination, thus overcoming the complexities of the soil matrix.
3. Bioremediation and mushrooms Normally, reference to bioremediation of toxic wastes implies the use of bacteria or lower fungi and rarely is the use of fleshy fungi (mushrooms) considered. However, higher fungi should not be forgotten just because they produce fruit bodies. Saprophytic mushrooms are natural decomposers because they secrete enzymes and acids that degrade organic polymers into simpler moieties. The dominion of mushroom mycelium is enormous since each colony extends long chains of cells forming a network that can occupy many square meters. Even the most popular edible mushroom (Agaricus bisporus) is capable of accumulating silver as demonstrated by Falandysz et al. [24], who investigated the uptake of this element from artificially enriched substrates. Accumulations of up to 150 mg/kg (on a dry weight basis) of silver were found in the fruit bodies (caps and stalks) when the level of the metal in the substrate was more than 12 times lower, showing that A. bisporus is not only an extractor but is also an efficient concentrator for this element. Silver (as silver nitrate) at this concentration did not affect mycelia growth or the emergence of fruiting bodies. In normal terrestrial plants the accumulation of silver is typically of the order of 60 mg/kg. Weber and coworkers [25] showed that the Basidiomycete Boletus badius is particularly efficient in accumulating gold and arsenic, which are stored in different parts of the mushrooms. Thus, gold is accumulated in caps and stalks, while arsenic accumulates in the hymenium. The amount of gold accumulated in the mushroom can be as high as 0.23 mg/kg, although it is estimated that for a plant to be defined as an hyperaccumulator it would probably store 1 mg/kg of this particular element [26]; in contrast, a terrestrial plant will accumulate typically only 5 mg/kg of gold and there is clearly a possibility of utilizing this fungus in the area of bioprospecting if not in biomining itself.
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The ability of mushrooms to concentrate toxic metals such as arsenic and cadmium is clearly taxonomically related. Vetter [27] investigated 88 species of the class Basidiomycetes. Significant accumulation of arsenic was found in species of Agaricus and in familyrelated fungi, such as Macrolepiota rhacodes, Flammulina velutipes, Lepista nebularis, and Clitocybe inversa. The highest concentration of cadmium (35 mg/kg) was also found within the genus Agaricus, although in contrast, the best cadmium-hyperaccumulating plant Thlaspi caerulescens can accumulate over 100 mg/kg [28]. Although the accumulation of arsenic is rarely observed in fungi (most accumulating only a few mg/kg), some species appear to be particularly adept at both storing and transforming this element. Stijve [29] investigated 300 species of mushroom and found that only six species showed abnormally high arsenic levels, but such levels ranged from 10 mg/kg up to 2.4 g/kg. This unusual accumulation is even higher than that found in some aquatic plants, such as Ceratophyllum demersum and Lagarosiphon major, which store up to 1.2 g/kg arsenic [30]. These plants grow in the Waikato river (New Zealand), which has high arsenic levels due to geothermal activity in the area. There are no terrestrial plants known to hyperaccumulate arsenic. In some of the investigated fungal accumulators (e.g. Entoloma lividum) arsenic was stored as the toxic arsenite and arsenate, but in others, inorganic arsenic was converted into organic derivatives of significantly lower toxicity, such as monomethylarsonic acid (Arsenicum coronaria), dimethylarsinic acid (Laccaria amethystina), and arsenobetaine (Sarcodom imbricatus and two Agaricus species), the last compound being a harmless metabolite that also occurs in sea fish and crustaceans. Stijve [29] emphasizes the fascinating similarity between the metabolism of arsenic in fungi and in mammals and marine fish. In comparison with our knowledge of the remediation of toxic metals by fleshy fungi, considerably less is known about the elimination of organic pollutants by these organisms, and this mirrors the situation with respect to higher plants. However, some strains of the highly appreciated gourmet mushroom Lentinus edodes (Shiitake) have been shown to possess the capacity to remove more than 60% of contaminating pentachlorophenol (PCP) from soil and convert it into pentachloroanisole [31], among other products. In subsequent work, the same group of workers [32] showed that monocultures of this mushroom were more efficient at removing PCP than were mixed cultures containing microflora with which this mushroom normally competes. Furthermore, after 10 weeks growth on contaminated soil, 99% of PCP was biotransformed by the monoculture and pentachloroanisole had been eliminated by this time; in contrast, only 42% PCP was metabolized by mixed cultures and pentachloroanisole was still present. Interestingly, phenol oxidase and Mn-peroxidase activities were much higher in the monoculture than in the mixed cultures. The ability to absorb and degrade chlorophenols appears to be characteristic of many Basidiomycetes. Thus, in addition to L. edodes, mycelia of fungi such as Armillaria, Ganoderma, Pleurotus, Polyporus, Coprinus, and Volvariella were all able to remove PCP when grown in a batch cultivation system [33]. The capacities of the mushrooms to absorb and degrade the contaminant varied considerably: mycelia of Polyporus was best at absorbing PCP (31 g/kg), while Armillaria showed the highest capacity to break down the compound (13 g/kg). Waste waters polluted with residues from the processes of olive oil extraction are regarded as environmental pollutants due to their high content of phenolic compounds, which are both
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antimicrobial and phytotoxic. Although these toxic wastes can be treated through microbial biodegradation, the use of other biosystems, such as the edible mushrooms, may show distinct advantages. Thus the oyster mushroom Pleurotus ostreatus was able to remove up to 90% of the phenol content of a solid state bioreactor supplemented with olive milling wastes (OMW) [34]. Zervakis et al. [35] found that the tolerance level of two Pleurotus species (P. eryngii and P. pulmonarius) towards raw OMW ranged from 25 to 50%. Other solid wastes, such as sugar-cane trash and bagasse as well as cork residues, may also present an environmental problem because of their toxic content and/or complex biological degradation, and these also are amenable to remediation using edible mushroom species, such as Pleurotus [36] and Lentinus and Ganoderma [37], respectively.
4. Conclusions In the last decade, hairy roots have helped markedly with the study of secondary metabolism, and their use continues to contribute to the understanding of biosynthesis, storage, and turnover of plant products. Based on our knowledge of the use and applications of hairy root cultures in this area, we believe that these extraordinary systems can contribute to phytoremediation research. Although vegetation-based bioremediation of contaminated sites has been shown to be advantageous in many situations, the full potential of this technology can be exploited only if solutions to the many remaining challenges can be found. Within this context, Agrobacterium-transformed roots can be used for screening plants suitable for cleanup of inorganic and organic pollutants, for the study of the potential pathways by which toxic compounds are broken down, and to determine the best ways in which the rhizosphere can be manipulated to achieve this purpose. Furthermore, the root zone, since it is the part of the plant in intimate contact with the contaminant, must be targeted for the expression of foreign genes with a view towards enhancing the uptake, bioaccumulation, or biotransformation of specific compounds: hairy roots are uniquely suitable as model systems for this purpose. Alongside roots of higher plants, the subterranean complex of mycelia associated with mushroom growth would appear to offer a number of possibilities in the field of remediation. There has been little research in this area, but the reported high efficiencies with which mushrooms can colonize areas of contaminated substrate and uptake and degrade exogenous toxins suggests that there is considerable potential in this approach. Liquid culture systems of mycelia also appear to be excellent model systems with which to commence screening and biochemical studies.
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[4] Glass DJ. The 1998 United States Market for Phytoremediation. D. Glass Associates, Needham, 1998. [5] Hooykaas PJJ, Beijersbergen AGM. The virulence system of Agrobacterium tumefaciens. Ann Rev Phytopathol 1994;32:157–79. [6] Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel B. Characterization of competent cells and early events in Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 1992;188:439–56. [7] Kathen A de, Jacobsen H-J. Cell competence for Agrobacterium-mediated DNA transfer in Pisum sativum L. Transgen Res 1995;4:184–91. [8] Hood EE, Gelvin SB, Melchen LS, Hoekema A. New Agrobacterium helper plasmid for gene transfer to plants. Transgen Res 1993;2:208–21. [9] Conner AJ, Dommisse EM. Monocotyledonous plants as hosts for Agrobacterium. Int J Plant Sci 1992;153:550–5. [10] Wilmink A, Van de Ven BCE, Dons JJM. Expression of the gus gene in the monocot tulip after introduction by particle bombardment and Agrobacterium. Plant Cell Rep 1992;11:76–80. [11] Mihalijevic S, Stipkovic S, Jelaska S. Increase of root induction in Pinus nigra explants using agrobacteria. Plant Cell Rep 1996;15:610–4. [12] Tzfira T, Yarnitzky O, Vainstein A, Altemen A. Agrobacterium rhizogenes-mediated DNA transfer in Pinus halepensis Mill. Plant Cell Rep 1996;16:26–31. [13] Kodama H, Irifune K, Kamada H, Morikawa H. Transgenic roots produced by introducing Ri-rol genes into cucumber cotyledons by particle bombardment. Transgen Res 1993;2:147–52. [14] Doran P, Nedelkoska TV. Uptake of heavy metals by hairy roots of hyperaccumulating plant species. Abs. of 217th National Meeting of the ACS. Anaheim, California, USA, BIOT 045, March 1999. [15] Soudek P, Podlipna R, Lipavska H, Vanek T. Bioaccumulation of heavy metals by hairy root cultures of Armoracia rusticana L. Pharmaceut Pharmacol Lett 1998;8:57–60. [16] Raskin I. Plant genetic engineering may help with environmental cleanup. Proc Natl Acad Sci USA 1996;93:3164–6. [17] Mackova M, Macek T, Kucerova P, Burkhard J, Pazlarova J, Demnerova K. Degradation of polychlorinated biphenyls by hairy root culture of Solanum nigrum. Biotechnol Lett 1997;19:787–90. [18] Mackova M, Macek T, Ocenaskova J, Burkhard J, Demnerova K, Pazlarova J. Biodegradation of polychlorinated biphenyls by plant cells. Int Biodeterior Biodegrad 1997;39:317–25. [19] Hughes JB, Shanks J, Vanderford M, Lauritzen J, Bhadra R. Transformation of TNT by aquatic plants and plant tissue cultures. Environ Sci Technol 1997;31:266–71. [20] Rhodes MJC, Robins RJ, Hamill JD, Parr AJ, Hilton M, Walton NJ. Properties of transformed root cultures. In: Charlwood BV, Rhodes MJC, editors. Secondary Products from Plant Tissue Cultures. Oxford: Oxford Scientific Publications, 1990. pp. 201–25. [21] Araujo BS, Charlwood BV, Pletsch M. The use of Daucus carota hairy root systems in phytoremediation studies. Abs. of 2nd IUPAC International Conference on Biodiversity, Belo Horizonte, MG, Brazil, July 1999. p. 142. [22] Kim YH, Yoo YJ. Peroxidase production from carrot hairy root culture. Enz Microb Technol 1996;18:531–5. [23] Schnoor JL, Licht LA, McCutcheon SC, Wolfe NL, Carreira LH. Phytoremediation of organic and nutrient contaminants. Environ Sci Technol 1995;29:318A–23A. [24] Falandysz J, Bona H, Danisiewicz D. Silver uptake by Agaricus bisporus from an artificially enriched substrate. Zeit Lebensmit Forsch 1994;199:225–8. [25] Weber A, Lehrberger G, Morteani G. Gold and arsenic in mushrooms, mosses and needles: Biochemical aspects of a middle ages geogenic dumping site in Oberviechtach, northern Oberpfaelzer Wald. Geol Bavar 1997;229–50. [26] Brooks RR. Geobotany and hyperaccumulators. In: Brooks RR, editor. Plants that Hyperaccumulate Heavy Metals. Wallingford: CAB International, 1998. pp. 55–94. [27] Vetter J. Data on arsenic and cadmium contents of some common mushrooms. Toxicon 1994;32:11–5. [28] McGrath SP. Phytoextraction for soil remediation. In: Brooks RR, editor. Plants that Hyperaccumulate Heavy Metals. Wallingford: CAB International, 1998. pp. 261–87. [29] Stijve T. Arsenic in mushrooms. Coolia 1995;38:181–90.
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[30] Robinson B, Outred H, Brooks R, Kirkman J. The distribution and fate of arsenic in the Waikato River system, North Island, New Zealand. Chem Spec Bioavail 1995;7:89–96. [31] Okeke BC, Smith JE, Paterson A, Watson-Craik IA. Aerobic metabolism of pentachlorophenol by spent sawdust culture of Shiitake mushroom (Lentinus edodes) in soil. Biotechnol Lett 1993;15:1077–80. [32] Okeke BC, Paterson A, Smith JE, Watson-Craik IA. Comparative biotransformation of pentachlorophenol in soils by solid substrate cultures of Lentinus edodes. Appl Microbiol Biotechnol 1997;48:563–9. [33] Chiu SW, Ching ML, Fong KL, Moore D. Spent oyster mushroom substrate performs better than many mushroom mycelia in removing the biocide pentachlorophenol. Mycol Res 1998;102:1553–62. [34] Setti L, Maly S, Iacondini A, Spinozzi G, Pifferi PG. Biological treatment of olive milling waste waters by Pleurotus ostreatus. Annali Chim 1998;88:201–22. [35] Zervakis G, Yiatras P, Balis C. Edible mushrooms from olive oil mill wastes. Int Biodeterior Biodegrad 1996;38:237–43. [36] Pani BK, Panda SN, Das SD. Bioconversion of sugar cane crop wastes into food by oyster mushroom, Pleurotus sajor-caju. Crop Res 1998;15:297–9. [37] Riu H, Roig G, Sancho J. Production of carpophores of Lentinus edodes and Ganoderma lucidum grown on cork residues. Microbiol 1997;13:185–92. [38] Brooks RR. Phytoremediation by volatilisation. In: Brooks RR, editor. Plants that Hyperaccumulate Heavy Metals. Wallingford: CAB International, 1998. pp. 289–312. [39] Crowley DE, Alvey S, Gilbert ES. Rhizosphere ecology of xenobiotic-degrading microorganisms. In: Kruger EL, Anderson TA, Coats JR, editors. Phytoremediation of Soil and Water Contaminants. Washington: ACS, 1997. pp. 20–36.
Novel biotechnological approaches in environmental remediation research Marcia Pletsch*, Brancilene Santos de Araujo, Barry V. Charlwood Universidade Federal de Alagoas, CCEN, Departamento de Química, Campus Universitário, Tabuleiro dos Martins, 57072-970 Maceió, Alagoas, Brazil
Abstract Two novel approaches, the use of Agrobacterium-transformed plant roots and mycelia cultures of fungi, are considered as research tools in the study of the remediation of soil, groundwater, and biowastes. Transformed roots are excellent model systems for screening higher plants that are tolerant of various inorganic and organic pollutants, and for determining the role of the root matrix in the uptake and further metabolism of contaminants. Edible and/or medicinal fungi may also be natural environmental remediators. Liquid cultures of fungal mycelia are appropriate model systems with which to commence screening and biochemical studies in this under-researched area of biotransformation. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Phytoremediation; Bioremediation; Organic contaminants; Inorganic contaminants; Hairy roots; Mushrooms
1. Introduction Bioremediation and phytoremediation are terms used to describe those methodologies that employ living organisms to remove toxic compounds from the environment. While bioremediation refers to the use of lower organisms (bacteria and fungi) and phytoremediation refers to the use of plants, both technologies share the advantages of in situ application and environmental acceptability compared with most physicochemical strategies. Since the remediation approaches avoid having to transport the contaminated material to the site of treatment, large savings on the costs of handling, transportation, and storage of contaminated or waste materials may be achieved. Extensive reviews are available concerning the specific advantages, limitations and economics of bio- and phytoremediation [1–4]. The present article addresses two innovative as* Corresponding author. Tel: 155-82-2141386; fax: 155-82-2141615. E-mail address: [email protected] (M. Pletsch) 0734-9750/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0 7 3 4 - 9 7 5 0 ( 9 9 ) 0 0 0 2 8 -2
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pects of this subject: the application of Agrobacterium rhizogenes-transformed roots (hairy roots) in phytoremediation studies and the use of higher fungi (mushrooms) in bioremediation. Each one of these biological systems can contribute to our understanding of the mechanisms involved in the removal, containment, or decomposition of a wide variety of pollutants, particularly petroleum-based compounds, polychlorinated biphenols (PCBs), heavy metals, pesticide-related compounds, potentially toxic biowastes, and even radioactive contaminants. Despite the fact that transformed roots and mushroom mycelia are cytologically, morphologically, and biochemically diverse structures, in terms of remediation they carry out very similar functions. Both represent the hidden complex of a highly branched matrix of organized living cells, which is at the interface of the remediating organism and the contaminated substrate. Whatever is the fate of a contaminant in a plant or fungus or its surroundings, remediation always starts with uptake or release of active molecules by the roots and mycelia. 2. Phytoremediation and transformed roots Phytoremediation is being developed as an alternative technology for removing or, more accurately, reducing the concentration of toxic organic and inorganic pollutants in air, water, and soil. In recent years, an array of new terminology has emerged to designate the various processes involved in phytoremediation, such as phytoextraction, phytotransformation, phytovolatilization, phytostimulation, rhizofiltration, and phytostabilization, the principles of which are summarized in Table 1. Hairy roots have been obtained from numerous plant species following simple infection of sterile tissue with the soil-borne bacterium Agrobacterium rhizogenes. Most of the studies utilizing hairy roots have been concerned with the formation of secondary compounds of pharmaceutical value and during the past 15 years have provided a powerful tool for the elucidation of metabolic pathways, enzymatic steps, and regulation of secondary metabolism.
Table 1 Strategies in phytoremediation Type
Fate of contaminants
Reference
Phytoextraction
Absorption from the soil and accumulation into the roots (or other plant tissues) in an unchanged form for later harvesting (phytomining) Absorption and bioconversion (catabolism or anabolism) in the roots (or other plant tissues) Uptake and conversion into a volatile form that is released into the atmosphere Stimulation of microbial biodegradation by plant exudates Absorption from aqueous environments and subsequent disposal of root material Sequestration, lignification or humification in the soil
[28]
Phytotransformation Phytovolatilization Phytostimulation Rhizofiltration Phytostabilization
[23] [38] [39] [4] [2]
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However, the use of hairy roots is not only limited to the production of secondary products: the system can be applied to the improvement of rooting ability in recalcitrant species, production of useful plant proteins (commercially important enzymes, lectins, and ribosome inhibiting proteins), regeneration of transgenic plants exhibiting beneficial traits, and, more recently, phytoremediation. Hairy roots can contribute to research and development of methods for phytotransformation, phytostimulation, rhizofiltration, and even for phytoextraction where the contaminant is to be sequestered in root tissue. The specific advantages of using hairy roots as model systems are: (1) they may be obtained from a wide range of gymnosperms and angiosperms; (2) they grow fast in dilute, hormone-free media and in the absence of light and are hence rapid and cheap systems to implement; (3) they present genetic and biochemical stability similar to that of the mother plant; and (4) they are isolated systems that permit phytoremediation studies to be performed without the interference of the microbiota, or even of other plant organs. Regarding the host range of A. rhizogenes, it was thought for a number of years that within the angiosperms, only dicotyledonous plants were susceptible to Agrobacterium infection, while monocotyledonous plants were outside the host range of the bacterium. However, more recent advances in the understanding of the processes involved in the infection event (e.g. chemical signalling, organization of the virulence operon [5], physiological state of the explants, and their competence for transformation [6,7]) has led to both the construction of super-virulent strains of Agrobacterium [8] and more appropriate protocols for the transformation itself. Thus, many more monocotyledons now appear to be hosts for A. rhizogenes than previously thought, and this extends the boundaries for the application of transformed roots [9,10]. Even within the gymnosperms there are many successful examples of transformation with A. rhizogenes resulting in root formation and development (i.e. in various Pinus species [11,12]). A further possibility to increase the range of plants for which transformed roots may be obtained is provided by the demonstration by Kodama et al. [13] that hairy roots may be formed by the direct bombardment of plant material (in this case cotyledon tissues of Cucumis sativus) with a plasmid containing the rol genes of A. rhizogenes. The screening for high level accumulation of metals by hairy roots is a very practical operation because of the uncomplicated manner in which the experiments may be performed in the laboratory, under controlled conditions, thus eliminating the difficulties of conducting extensive trials in the field. That transformed roots retain the biochemical capacity of the parent plant is illustrated by the work of Doran and Nedelkoska [14], who showed that hairy roots of the well-known hyperaccumulating plants of the Cruciferae, namely Thlaspi caerulescens (which can contain over 3% zinc/cadmium) and species of Alyssum (which can accumulate up to 1% dry weight of nickel), retained their unusual ability to remove heavy metals from the culture medium. Extension of this type of search to discover other plants that might overaccumulate metals has led, for example, to the demonstration that hairy roots of horseradish (Armoracia rusticana) accumulated mercury, iron, and copper ions at high rates [15]. Apart from the elegant simplicity of using hairy roots for screening purposes, there is also the possibility of introducing new genes into roots through single-step transformations using genetically altered A. rhizogenes. The biochemical consequences promoted by the new gene product can then be analyzed directly in the transformed culture. Suitable target genes for such studies might be those encoding metal-binding proteins, such as metalloenzymes, met-
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allothioneins, metal storage proteins, and metal-activated enzymes, as well as those responsible for the synthesis of the phytochelatins, low molecular weight peptides with high affinities for metals [16]. Regarding organic contaminants, there is considerable evidence that demonstrates the feasibility of using transformed root cultures for the purpose of screening plants capable of degrading such pollutants and the subsequent identification of metabolic products. In one such example, Mackova et al. [17,18] showed that hairy root cultures of Armoracia rusticana, Solanum aviculare, Atropa belladona, and Solanum nigrum were able to degrade PCBs, with the last example presenting the highest metabolic capability, which lead to a reduction of PCB concentration in the medium from 100 to 40 ppm in 30 days. This efficient degradation of exogenous PCBs was associated with increased peroxidase activity in the root system. Furthermore, Hughes et al. [19] showed that the explosive 2,4,6-trinitrotoluene was converted by Catharanthus roseus hairy root cultures into minor products (i.e. aminated nitrotoluenes, and major, but uncharacterized, soluble products that were stored both within the root tissue and in the medium itself). Clearly it is very important to identify such catabolic products to determine the degree of toxicity in comparison with the original pollutant. Undoubtedly the hairy root system is very suitable for the determination of catabolic pathway reactions and their enzymatic regulation. Learning about the degradation pathways of specific organic pollutants in target plants is fundamental to the study of the overexpression of controlling genes and to the production of transgenic plants with enhanced degradative capabilities. Indeed, it is pointless to transfer a bacterial, fungal, or animal gene to a plant that would convert the contaminant into an intermediate that could not be stored or further transformed by the natural enzymatic machinery of that plant. When plants are subjected to physical or chemical stress or microbial or herbivore attack, their typical response involves the induction of either a new metabolic pathway or the augmentation of an existing pathway. Often, such elicited pathways lead to the formation of products (secondary metabolites) that form part of the plant defense mechanisms. Hairy roots have been a valuable tool in the study of the enzymatic regulation of such secondary pathways [20] and in the understanding of elicitor response mechanisms. Since pollutants constitute stressing agents to plants in general, the application of elicitation studies to hairy roots with the aim of determining which genes are triggered when tissues are exposed to specific contaminants will certainly be advantageous for phytoremediation technology. The early stages of xenobiotic exposure and its limiting factors (type and concentration of contaminant and length of exposure) must be investigated to understand how plants react in the face of chemical aggressors. We have investigated the effects of various concentrations of phenol, 2-chlorophenol, 2,6dichlorophenol, and 2,4,6-trichlorophenol on 30 clonal hairy root lines of Daucus carota [21]. The chlorinated derivatives were more toxic to the transformed carrot roots than phenol itself (i.e. root growth was possible in the presence of chlorophenols only at concentrations 20 times lower than that of phenol). The capacity of carrot roots to translocate phenols from the medium to the roots and further transform them was totally independent of the growth characteristics of the specific clones. This is an important point, since root clones may show distinctive biochemical attributes as reported for secondary compound accumulation. If hairy root cultures are to be used as model systems for screening tolerant plants capable of metab-
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olizing xenobiotics, then they must exhibit these properties independently of their origins (clonal or mixed cultures). Phenol and chlorinated phenols were able to elicit peroxidase activity by up to 100% in carrot hairy roots and this activity remained elevated, while the removal and metabolism of the major part of the phenols occurred [21]. It has also been shown [22] that pyrogallol, 2-aminophenol, p-cresol, and catechol elicit peroxidase activity in hairy root cultures of D. carota. Together with peroxidases, other enzymes of plant origin, such as dehalogenases, laccases, nitroreductases, and nitrilases, are reportedly involved in phytoremediation processes [23], some or all of which may be inducible by chemical stress. The situation concerning reductases, laccases, and dehalogenases in carrot roots is currently being investigated in our laboratory. Finally, hairy roots can be used to study root-microbe interactions and to understand how soil microorganisms are influenced by the presence of roots and/or their exudates. For example, the appropriateness of using plants to increase the activity of native or introduced microorganisms that can degrade xenobiotics (phytostimulation) and the conditions under which such a process may occur can readily be evaluated using the hairy root system. Experiments can be designed whereby roots may be inoculated with specific microorganisms, individually or in combination, thus overcoming the complexities of the soil matrix.
3. Bioremediation and mushrooms Normally, reference to bioremediation of toxic wastes implies the use of bacteria or lower fungi and rarely is the use of fleshy fungi (mushrooms) considered. However, higher fungi should not be forgotten just because they produce fruit bodies. Saprophytic mushrooms are natural decomposers because they secrete enzymes and acids that degrade organic polymers into simpler moieties. The dominion of mushroom mycelium is enormous since each colony extends long chains of cells forming a network that can occupy many square meters. Even the most popular edible mushroom (Agaricus bisporus) is capable of accumulating silver as demonstrated by Falandysz et al. [24], who investigated the uptake of this element from artificially enriched substrates. Accumulations of up to 150 mg/kg (on a dry weight basis) of silver were found in the fruit bodies (caps and stalks) when the level of the metal in the substrate was more than 12 times lower, showing that A. bisporus is not only an extractor but is also an efficient concentrator for this element. Silver (as silver nitrate) at this concentration did not affect mycelia growth or the emergence of fruiting bodies. In normal terrestrial plants the accumulation of silver is typically of the order of 60 mg/kg. Weber and coworkers [25] showed that the Basidiomycete Boletus badius is particularly efficient in accumulating gold and arsenic, which are stored in different parts of the mushrooms. Thus, gold is accumulated in caps and stalks, while arsenic accumulates in the hymenium. The amount of gold accumulated in the mushroom can be as high as 0.23 mg/kg, although it is estimated that for a plant to be defined as an hyperaccumulator it would probably store 1 mg/kg of this particular element [26]; in contrast, a terrestrial plant will accumulate typically only 5 mg/kg of gold and there is clearly a possibility of utilizing this fungus in the area of bioprospecting if not in biomining itself.
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The ability of mushrooms to concentrate toxic metals such as arsenic and cadmium is clearly taxonomically related. Vetter [27] investigated 88 species of the class Basidiomycetes. Significant accumulation of arsenic was found in species of Agaricus and in familyrelated fungi, such as Macrolepiota rhacodes, Flammulina velutipes, Lepista nebularis, and Clitocybe inversa. The highest concentration of cadmium (35 mg/kg) was also found within the genus Agaricus, although in contrast, the best cadmium-hyperaccumulating plant Thlaspi caerulescens can accumulate over 100 mg/kg [28]. Although the accumulation of arsenic is rarely observed in fungi (most accumulating only a few mg/kg), some species appear to be particularly adept at both storing and transforming this element. Stijve [29] investigated 300 species of mushroom and found that only six species showed abnormally high arsenic levels, but such levels ranged from 10 mg/kg up to 2.4 g/kg. This unusual accumulation is even higher than that found in some aquatic plants, such as Ceratophyllum demersum and Lagarosiphon major, which store up to 1.2 g/kg arsenic [30]. These plants grow in the Waikato river (New Zealand), which has high arsenic levels due to geothermal activity in the area. There are no terrestrial plants known to hyperaccumulate arsenic. In some of the investigated fungal accumulators (e.g. Entoloma lividum) arsenic was stored as the toxic arsenite and arsenate, but in others, inorganic arsenic was converted into organic derivatives of significantly lower toxicity, such as monomethylarsonic acid (Arsenicum coronaria), dimethylarsinic acid (Laccaria amethystina), and arsenobetaine (Sarcodom imbricatus and two Agaricus species), the last compound being a harmless metabolite that also occurs in sea fish and crustaceans. Stijve [29] emphasizes the fascinating similarity between the metabolism of arsenic in fungi and in mammals and marine fish. In comparison with our knowledge of the remediation of toxic metals by fleshy fungi, considerably less is known about the elimination of organic pollutants by these organisms, and this mirrors the situation with respect to higher plants. However, some strains of the highly appreciated gourmet mushroom Lentinus edodes (Shiitake) have been shown to possess the capacity to remove more than 60% of contaminating pentachlorophenol (PCP) from soil and convert it into pentachloroanisole [31], among other products. In subsequent work, the same group of workers [32] showed that monocultures of this mushroom were more efficient at removing PCP than were mixed cultures containing microflora with which this mushroom normally competes. Furthermore, after 10 weeks growth on contaminated soil, 99% of PCP was biotransformed by the monoculture and pentachloroanisole had been eliminated by this time; in contrast, only 42% PCP was metabolized by mixed cultures and pentachloroanisole was still present. Interestingly, phenol oxidase and Mn-peroxidase activities were much higher in the monoculture than in the mixed cultures. The ability to absorb and degrade chlorophenols appears to be characteristic of many Basidiomycetes. Thus, in addition to L. edodes, mycelia of fungi such as Armillaria, Ganoderma, Pleurotus, Polyporus, Coprinus, and Volvariella were all able to remove PCP when grown in a batch cultivation system [33]. The capacities of the mushrooms to absorb and degrade the contaminant varied considerably: mycelia of Polyporus was best at absorbing PCP (31 g/kg), while Armillaria showed the highest capacity to break down the compound (13 g/kg). Waste waters polluted with residues from the processes of olive oil extraction are regarded as environmental pollutants due to their high content of phenolic compounds, which are both
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antimicrobial and phytotoxic. Although these toxic wastes can be treated through microbial biodegradation, the use of other biosystems, such as the edible mushrooms, may show distinct advantages. Thus the oyster mushroom Pleurotus ostreatus was able to remove up to 90% of the phenol content of a solid state bioreactor supplemented with olive milling wastes (OMW) [34]. Zervakis et al. [35] found that the tolerance level of two Pleurotus species (P. eryngii and P. pulmonarius) towards raw OMW ranged from 25 to 50%. Other solid wastes, such as sugar-cane trash and bagasse as well as cork residues, may also present an environmental problem because of their toxic content and/or complex biological degradation, and these also are amenable to remediation using edible mushroom species, such as Pleurotus [36] and Lentinus and Ganoderma [37], respectively.
4. Conclusions In the last decade, hairy roots have helped markedly with the study of secondary metabolism, and their use continues to contribute to the understanding of biosynthesis, storage, and turnover of plant products. Based on our knowledge of the use and applications of hairy root cultures in this area, we believe that these extraordinary systems can contribute to phytoremediation research. Although vegetation-based bioremediation of contaminated sites has been shown to be advantageous in many situations, the full potential of this technology can be exploited only if solutions to the many remaining challenges can be found. Within this context, Agrobacterium-transformed roots can be used for screening plants suitable for cleanup of inorganic and organic pollutants, for the study of the potential pathways by which toxic compounds are broken down, and to determine the best ways in which the rhizosphere can be manipulated to achieve this purpose. Furthermore, the root zone, since it is the part of the plant in intimate contact with the contaminant, must be targeted for the expression of foreign genes with a view towards enhancing the uptake, bioaccumulation, or biotransformation of specific compounds: hairy roots are uniquely suitable as model systems for this purpose. Alongside roots of higher plants, the subterranean complex of mycelia associated with mushroom growth would appear to offer a number of possibilities in the field of remediation. There has been little research in this area, but the reported high efficiencies with which mushrooms can colonize areas of contaminated substrate and uptake and degrade exogenous toxins suggests that there is considerable potential in this approach. Liquid culture systems of mycelia also appear to be excellent model systems with which to commence screening and biochemical studies.
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