Lignin in crop cultivation and bioremediation

Environmental Engineering and Management Journal, September 2005, Vol. 4, No. 3, 273-292

http://omicron. ch. tuiasi. ro/EEMJ/

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LIGNIN IN CROP CULTIVATIONS AND BIOREMEDIATION

Alfred Abaecherli1, Valentin I. Popa2∗

1International Lignin Institute,Rue du Grand-Chene 5,Ch-1003 Lausanne, Switzerland

2 Technical University of Iasi, 71 Mangeron Blvd., Iasi-700050, Romania

Abstract It is known that in the natural conditions of organic carbon cycle lignins represents the main source of humus formation. This complex transformation could be accelerated by chemical and biochemical modification of lignins. Along with this natural aromatic polymer, the polyphenols can be considered. Based on chemical and biochemical modification of lignins and polyphenols and the properties of resulted products they can be used to improve soil fertility and to perform different kind of bioremediations. In this review the main possibilities to manipulate these processes are presented. Keywords: lignins, polyphenols, nonconventional fertilizers, slow release agents, growth stimulators, sequestering agents, soil conditioners, compost activators, bioremediation 1. Introduction

The lignins are biosynthesized in plants to carry out different functions,

such as storage of energy, bonding agent between plant cells, protection agent against microorganisms, antioxidant, and hydrophobic agent. Lignin is an important precursor for stabilized soil organic carbon. Terrestrial ecosystems that produce large amounts of lignin are thought to have high potential for sequestering carbon which can decrease global warming by reducing atmospheric CO2. After the death of the plants, their compounds are transformed at the soil level with humus formation. Based on biological transformation some utilisations of lignins could be developed among other things being crops cultivation and bioremediation.

Bioremediation by definition is concerned with fate and, if necessary, removal of unwanted organic chemicals from soil. At present, bioremediation is considered as a less expensive alternative to physical and chemical means of degradation of organic pollutants. It deals, with substances that are anthropogenic distributed in nature and recalcitrant. At the same time it is not lacked of interest to involve the bioremediation to improve some properties of the soils. ∗ Author to whom all correspondence should be addressed: e-mail: vipopa@ch. tuiasi. ro

“Gh. Asachi” Technical University of Iasi, Romania

Abaecherli and Popa/Environmental Engineering and Management Journal 4 (2005), 3, 273-292

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2. Formation of soil organic matter The ultimate model cycling of the carbon moiety of chemicals in nature

is terrestrial carbon cycle. Carbon dioxide and water are used for the synthesis of phytomass which, in turn, is mineralized by microorganisms and animals to carbon dioxide and water again, the whole cycle being driven by energy from sun light (Fig. 1).

Fig. 1. The progress of humus formation resulting from microbial degradation of plants (Christman and Ogleshy, 1971)

In this process the polysaccharides from phytomass are used by

microorganisms as carbon sources, while it is known that by the microbiological action, lignin from vegetal wastes is transformed at soil level in organic products with physiological activity on plants development. Thus, lignin may be considered as an initial source for humic acids formation which represents a main component of the soil humus (Fig. 2).

On the other hand, some micromolecular compounds resulted from plant wastes decomposition, along with polyphenols coming from extraction of plant residues could be inhibitors or promoters of plants growth. Therefore, the knowledge of complex processes including soil, plants and microorganisms allow us to use the lignin and its derivatives to control both soil fertility and plants development with a view to increase soil productivity and to perform soil bioremediation.

Returned in the atmosphere

Lignin 20%

Lignin Humus

Ash,Fat,Protein10%

Cellulose

Starches, Pectins, Gums

Sugars 10%

Microbial bodies

CO2 CO2

CO2 CO2

Reused by plants

start after a week after a month after a year

Returned in the atmosphere

Lignin 20%

Lignin Humus

Ash,Fat,Protein10%

Cellulose

Starches, Pectins, Gums

Sugars 10%

Microbial bodies

CO2 CO2

CO2 CO2

Reused by plants

start after a week after a month after a year

Lignin in crop cultivations and bioremediation

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Fig. 2. The pathway of humus formation Several pathways are proposed for the formation of humic substances

during the decay of plant and animal remains in soil, the main ones being shown in the Fig. 3.

The classical theory is that these substances represent modified lignins (pathway 1), but the majority of present-day of investigators favor a mechanism involving quinones (pathways 2 and 3). In practice all four pathways must be considered as likely mechanisms for the synthesis of humic and fulvic acids in nature, including sugar-amine condensation (pathway 4). These four pathways may operate in all soils, but not to the same extent or in the same order of importance. A lignin pathway may predominate in poorly drained soils and wet sediments (swamps, etc), whereas synthesis from polyphenols may be of considerable importance in certain forests soils. The frequent and sharp fluctuations in temperature, moisture and irradiation in terrestrial surface soils under a harsh continental climate may favor humus synthesis by sugar-amine condensation.

For many years it was thought that humic substances were derived from lignin (pathway 1). According to this theory, lignins are incompletely utilized by microorganisms and the residue becomes part of the soil humus.

Living organisms

Lignins and phenols resulted from the transformation of organic

wastes Phenols from

microbial synthesis

Degradation of proteins

Monomers and decomposition products

Depolymerization Demethylation Carbon and nitrogen sources for

microorganism Direct microbial synthesis

Different phenolic intermediates Final products of energetic metabolism,

CO2,H2O,NH 3 and other organic compounds Oxidation degradation of side chains –

formation of carboxylic aldehyde groups

Mono, di - and triphenols , benzoic acids, phenolicpolymers

Enzymatic oxidation, self oxidation

Free phenolic radicals, hidroxyquinones

Oxidative splitting of the aromatic ring

Direct microbial synthesis

Humic substances condensation

Integration in different circuits in the soil

Consumption by plants

Decomposition and biological transformation of dead organisms and organic wastes including

all types of manures


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Fig. 3. Mechanisms proposed for the formation of soil humic substances (web

page, h) Modifications in lignin include loss of methoxyl (OCH3) groups with

generation of hydroxyphenol and oxidation of aliphatic chains to form COOH groups. The modified material is subject to further unknown changes to yield first humic acids and then fulvic acids (Fig. 4).

On the other hand phenolic acids released from lignin during microbiological attack undergo enzymatic conversion to quinones which polymerize in the presence or absence of amino compounds to form humic like macromolecules (pathways 2 and 3). Another theory (pathway 4) considers that reducing sugars and amino acids, formed as by-products of microbial metabolism, undergo nonenzymatic polymerization to form brown nitrogenous polymers of the type produced during dehydration of certain food products at moderate temperature.

A major objection to this theory is that the reaction proceeds rather slowly at the temperatures found under normal soil conditions. However, drastic and frequent changes in the soil environment (freezing and thawing, wetting and drying), together with the intermixing of reactants with mineral material having catalytic properties may facilitate condensation. An attractive feature of the theory is that the reactants (sugars, amino acids etc.) are produced in abundance through the activities of microorganisms.

Plant residues

Transformation by microorganisms Modified lignins

Polyphenols Sugars

Quinones

Lignin decomposition products

Humic substances

2 34 1

Amino compounds

Quinones


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Fig. 4. Mechanism proposed for lignin participation in humus formation These transformations could be accelerated by the chemical

modification of lignins. In these conditions, the lignins or their derivatives could be considered as being nonconventional additives in the crops cultivation and bioremediation, because these compounds have an important role in:

- the energy management and distribution (regulation of carbon cycle) - the management of chemical nutrients - the maintaining the trophic web - the soil structuring - the sequestration of pollutants (e. g. bind heavy metal ions,

solubilize petroleum hydrocarbons etc. ) Also, they must have in mind that lignins may be byproducts of pulp and

paper industry, are inexpensive and readily available, are non-toxic and they can be converted into water soluble bioactive compounds.

3. Utilization of lignins and their derivatives in the crops cultivation and bioremediation

Having in mind the aspects above mentioned, the lignins and their

derivatives could carry out the following functions: 1. soil conditioners (in natural state as fertilizer; for the improvement of

salty soil)

Lignin

Attack by microorganisms

Lignin building units

Further utilization by microorganisms

Residium

Demethylation, oxidation and condensation with N compounds

Humic acids

Fulvinic acid

Fragmentation to smaller molecules

Lignin

Attack by microorganisms

Lignin building units

Further utilization by microorganisms

Residium

Demethylation, oxidation and condensation with N compounds

Humic acids

Fulvinic acid

Fragmentation to smaller molecules


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2. mineral nutrient sources used in a nonconventional manner (i. e. low rate; as ammonized/oxyammonolyzed lignin, as lignostimulator complex, as support for nitrogen containing fertilizers)

3. wetting agents or surfactants 4. biological inoculants and activators (as compost based on lignin) 5. plant stimulants and growth regulators ( e. g. modified by oxidative

nitration-nitroquinone polycarboxylic acids) 6. nonconventional fertility concepts or programms 7. as soil structuring agent and bioremediation of polluted soils

The surveillance of literature data evidenced some possibilities to use non- or modified lignins in the directions presented in Table 1, some of them being discussed in the following. 3. 1. Nonconventional fertilizers

If the lignosulfonates are used in agriculture the higher efficiency was

observed in the case of ammonium lignosulfonates resulted in the sulfite delignification of wood with ammonioum base or by exchaniging of cation with ammonium ion. At the same content of nitrogen, ammonium lignosulfonates have proved the same, or sometimes higher activity, as ammonium sulfate. The nitrogen content of ammonium lignosulfonates is about 3-4 % of mass of dried product. For a nitrogen dose of 50 kg/ha, it is necessary to use 1.5 t/ha lignosulfonates. The experiments carried out with ammonium lignosulfonates with a nitrogen content of 3.4 - 3.8 % provided from a sulfite pulp mill from Romania (closed at present) and with lignin from flax delignification (Granit type pF 30-35 flax soda pulping) have confirmed the efficiency of both products at dosage above mentioned (Dumitru et al. , 2003, a, b). The researches have been focused on the conversion of technical lignins to long term fertilizers. The investigations have shown that technical lignins from various pulping process differ as to their reactivity; concerning N-incorporation, the reactivity decreases in the following order: lignosulfonate, kraft lignin, organocell lignin, kraft lignin (CO2), ASAM lignin (Fischer and Schiene, 2002). Concerning the aspects of plant nutrition it was supposed that the nitrogen incorporated is hydrolysable and plant-available to various extents. Being organically bounded it exists both in the lower- and in the higher molecular (>10,000) mass fractions of N-lignin.

Low-molecular mass phenolic acids may be formed in analogy with natural lignin degradation. Phytophysiological effects such as the inhibition of nitrification suggest the existence of quinoid structures.

In pot experiments of forest plants, N-lignins from conventional spent sulfite liquors and prepared using oxidative ammonolysis process were found to contain physiologically active substances which temporarily seemed to have a negative effect on plant growth. These influences are attributable to substances in the lower molecular mass range. The effects are obviously connected to products that may be formed from residual carbohydrates in the spent sulfite liquors.


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Table 1. Possibilities of lignins and polyphenols utilization in the fields of crops cultivation and bioremediation

Utilizations

Lignins/lignin derivatives

Nonconventional fertilizers

Lignosulfonates, ammoniated lignosulfonates and hydrolysis lignin, oxiammonolyzed lignosulfonates and hydrolysis lignin [fertilizers with N-slow release, granulation (bonding agents), micronutrients, formulation, chelating agents (microelements )], oxidated lignins: lignosulfonates, hydrolysis lignin, kraft lignin [using O2 (air)/ammonia with/without catalysts,H2O2, persulfates] (Abduazimov et al., 1979; Arkhipov et al., 1992; Deineko and Blain, 1981; Detroyt, 1988; Fischer and Schiene, 2002; Ishibashi et al., 1977; Joffe et al., 1979; Kazarnovskii et al., 1976; Kimura, 1983; Kin et al., 1974; Martyushek et al., 1978; Mozheiko et al., 1987; Telysheva and Pankova, 1978; Zakis and Nieberte, 1973a; Zakis et al, 1973b; web pages a, b, c, d, e, f, g)

Slow release for pesticides, insecticides, herbicides

Crosslinked lignin with epichlorhydrine, kraft lignin, alcell lignin, lignosulfonates (non- or modified), sulfonated kraft lignin, alkali lignin (Dellicoli and Dilling, 1976a; Dellicoli et al., 1976b; Riggle, 1985; Westvaco Corp., 1976; Wilkins, 1983)

Growth stimulators

Aqueous soluble lignin,oxidated lignins (oxyammonolysis of lignosulfonates, hydrolysis lignin), nitration or oxidative nitration of hydrolysis lignin, lignocellulose, ammoniated nitrolignin, condensed lignosulfonates with urea, polyphenols (web page h; Bobu et al., 1991; Chudakov et al., 1965; Egorov et al., 1973; Egorov et al., 1975; Egorov et al., 1978; Hattenschwiler and Vitousek, 2000; Kaplunova et al., 1985; Komisarov et al., 1965; Popa and Popa, 1992; Popa and Beleca, 1994; Popa et al, 1995; Popa et al., 2000; Popa et al., 2002; Tudose and Popa, 2000)

Seed coatings

Lignosulfonates (Na, Ca), lignosulfonates sugar free, desulfonated lignosulfonates (web page f; Jinxia, 1991)

Sequestering agents Hydrolysis lignin,lignin and lignin-derivatives (Balogh et al, 1978; Keane and Ghoshal, 2001; Tisch et al, 2003)

Soil conditioners

Kraft lignin,oxidized kraft lignin, grafted lignosulfonates with acrylic/methacrylic acids or acrylonitrile, composted hydrolysis lignin (HL) or amonized HL, lignosulfonates (NH4

+, Na+), alkali treated lignin followed by activation with CuO (Builov et al., 1979; Couz et al., 1991; Danilova and Plastkov, 1980; Feodorets et al., 1983; Ikramov and Antonov, 1979; Khitrina et al., 1980; Kislenko, 1998; Kyoko and Gyosuke, 2002; Pesnyakevich et al., 1980; Sokolova et al., 1975; Zaslavsky and Rozenberg, 1977)

Wetting and surfacting agents

Kraft lignin highly sulfonated (Kraftsperse 25 M; Reax 88 A,88 B, and 100 M) kraft lignin low- to mid-level sulfonated (Reax 83 A and 85 B) (web page b), lignosulfonates from softwood ( Diwatex S-5, Ulfoxane, Ultrazine) (web pages d, f), lignosulfonates from hardwood (web page e)

Compost activators

Hydrolysis lignin, wastes from pulp and paper industry (Builov et al., 1979; Couz et al., 1991; Danilova and Plastkov, 1980; Feodorets et al., 1983; Ikramov and Akhatov, 1979; Khitrina et al., 1980; Kislenko, 1998; Krivulin et al., 1979; Kyoko and Gyosuke, 2002; Pesnyakevich et al., 1980; Sokolova et al, 1975; Zaslavsky and Rozenberg, 1977)

Bioremediation Hydrolysis lignin, lignosulfonates, alkali lignin, polyphenols (Huttermann and Majcherzyk, 1998; ; Liu et al, 1999; Sung-Cheol et al., 2004; Zohar and Yitzak, 1988)

Plant immunology Polyphenols (Boyce and Bernett, 1998)


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The process of oxidative ammonolysis is to be controlled in a way that the formed heterocyclic structures are humus-like incorporated in the macromolecular organic matrix.

At soil level the high molecular mass lignins are degraded into small fractions by reactions with reactive oxygen species in aerobic process due to microbial attack according to radical mechanisms. In this connection, the C and OCH3 contents are reduced while the N and O contents as well as the contents of OH, C=O and COOH groups increase. Reactive o-diphenol structures and o-quinoid structures are formed.

The similarity of basic reactions between oxidative ammonolysis and the natural humification permits a discussion of analogies despite distinct differences. Oxidative ammonolysis in vitro employs harsher conditions that drastically shorten the reaction time in contrast to the humification process that takes place in nature (Fig. 5).

Fig. 5. Reaction scheme for artificial humification of lignin (sob N = not

readily hydrolysable nitrogen) (Fischer and Schiene, 2002) The biogenic organic matter is converted in nature. In vitro, however,

the parent material is technical lignin which has already been altered during pulping and contains only low quantities of admixed substances. The particularly favorable effects of artificial humus substitutes (N-lignin) are shown in their abilities to function as excellent organic fertilizers. They are active over several growing seasons analogous to the natural humus containing various types of N-bonds. Those N-bonds, specifically, are: (1) ammonium – N which is immediately available for the plants; (2) medium term, plant available amide – N and (3) long-term, plant – available, organically bonded – N.

The artificial humus substitutes also improve the nutrient relations and

Technical lignins

Artficial humus N modified products NH4

+ -CONH2 sob N

Demethoxylation/demethylation Formation of o-quinones Oxidation of aliphatic side chain Formation of new C-C bonds Aromatic link cleavage Reaction and condensation with N

O2

NH3

Oxidative ammonolysis NH3,O2, air T,p


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promote soil-microbial processes in the near surface soil substrate layers, resulting in favorable conditions for plant growth and a high productive evaporation. The latter, coupled with the time-phased N release from the various types of bonds, leads to a markedly lower leaching as compared to mineral fertilizers.

3. 2. Slow release agents for pesticides, insecticides and herbicides

On large scale non-modified lignosulfonates (Na+, NH4

+), unsulfonated and sulfonated kraft lignin are used in the field of agrochemicals and bioremediation. Thus, the following directions could be mentioned: dispersants, agents for controlled release of pesticides, insecticides or herbicides, fertilizers formulation (granulation of nutrients - ureea, inorganic fertilizers, chelation of micronutrients-boron, chlorine, cobalt, copper, iron, manganese, molibdenum, sodium and zinc, in composition along with compounds which stimulates different stages in plant propagation).

In agricultural lands it might not be appreciated if a pesticides that is added to soil disappears to soon. To avoid this phenomenon, either crop rotation can be applied, implying the use of different pesticides with each different crop or special extenders (e. g. based on lignin derivatives) applied that protect the pesticides against degradation.

Other field trials are reported in the former Soviet Union for hydrolysis lignin using large quantities of nonmodified (as chelating agent for micronutrients, as mixture with inorganic fertilizers or soil conditioner) or in modified form by composting, ammonization and oxyammonolysis.

3. 3. Growth stimulators

The obtained results along with literature information could recommend

the utilization of lignosulfonates, lignin and polyphenols as growth stimulators to improve the soil fertility and for bioremediation of polluted area (e. g. with heavy metals, hydrocarbons etc. ). In these conditions an effect of rhizosphere is expected. The rhizosphere is a unique and dynamic zone of interaction between roots of plants and soil microorganisms. This region is characterized by the presence of a biomass and an increased soil activity. The community of rhisosphere is composed from microbios (bacteria, fungi and algae) and micro- and mezofauna (protozoa, nematodes, insects). The abundance of the microorganisms in the rhizosphere is of 5 till 20 or even 100 times higher than in the uncultivated soils. The symbiosis between plants and microorganisms can assures the protection of the plants against of toxic compounds along with their biodegradation (Curl and Truelove, 1986).

The fixation of nitrogen by plants becomes possible along with the formation of polyphenolic compounds from biodegradation of lignin. The polyphenols could represent signal molecules which can induce phytohormonal exchanges (Fig. 6), influencing some modifications at the level of root cortical


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cells. In this way the division and differentiation of cell are starting.

Fig. 6. Influence of bioregulators on essential physiological processes in plants (Popa and Beleca, 1994)

It is known that soil microorganisms degrade the lignin and their

derivatives by cleaving off units consisting of one, two or most three phenolic acid moieties. These transformations are accelerated when the modified lignins are used, especially in the case of those with a high degree of degradation. On the other hand some chemicals from plants may be released from living leaves as volatiles or leachates, or from roots through exudation or sloughing of dead tissue. They also may leach from litter on the soil surface. Most of them are of polyphenolic nature. These chemicals compounds influence the biological processes in a complex manner both at the soil level (in the humus formation) and plant development (by their intervention in the hormonal chain). Thus, in the last case it was possible to identify products with inhibitory (e. g. herbicides or pesticides) or with stimulatory action on different stages of plants propagation (seeds germination, plant grafting, plant developments a. s. o. ). Also, among them the compounds that can act on the expression of heredity program in another plant were identified.

Polyphenols are the most widely distributed class of plant secondary metabolites and several thousands different compounds have been identified. Polyphenols play many different roles in plant biology and human life, including UV protective agents, defensive compounds against herbivores and pathogens, associated with adventitious root production, contributors to plant colors, to the taste of food and drink and pharmaceuticals. For example, polyphenols extracted from several eucalyptus species are related to their disease resistance (Boyce and Bernett, 1998).


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In the soil, soluble polyphenols face four different fates. They might be degraded and mineralized as carbon source by heterotrophic microorganisms; they can be transformed into insoluble and recalcitrant humic substances by polymerization and condensation reactions (with the contribution of soil microorganisms); they might adsorb to clay minerals or form chelates with Al or Fe ions; or they might remain in dissolved form, leached by percolating water, and finally leave the ecosystem as part of dissolved organic carbon.

There is evidence for both negative and positive effects of phenolic compounds on the activity of soil organisms and hence on decomposition and nutrients mineralization. The regulation of nutrient mineralization through soil microbial activity is only one way that polyphenols can influence nutrient cycling. Complexation with inorganic sesquioxides, cations and proteins might also allow sustained P and base cation availability in highly weathered soils, and an advantage to the plant-mycorrhiza symbiosis over free-living microorganisms in the competition for N.

3. 4. Soil conditioning, fertilizers and sequestering agents

The lignin and its derivatives can be used to obtain soil conditioners. Thus, hydrolysis lignin was applied as conditioning agent (30 t/ha) with the aim to ameliorate acid steppe soils. It was observed the modification of the equilibrium of sodium and magnesium ions along with an increasing of crop productivity (Ikramov and Akhatov, 1979). In other experiments soluble fractions of lignin obtained by treatment with nitric acid (10 %) or nitric acid together with KOH, ammoniated nitrolignin or sulfolignin (Couz et al., 1991), activated lignin by alkali and CuO (Khitrina et al. , 1980), and lignosulfonates were used. These products improve the structure of the soil and its fertility. Kraft lignin was modified with sodium hydroxide and urotropine and a soil conditioner was obtained, which can be used on a large variety of soils (Sokolova et al., 1975).

Soil conditioners were prepared by the modification of water soluble lignin derivatives (lignosulfonates and nitrolignin) with acrylic monomers. The obtained preparations increase the content of water stable aggregates with the size more than 0. 25 mm to 90 %, as well as they improve water and gas exchange in soil. The soil cultivated in the presence of these preparations (75 –150 kg/ha) resists to water and air erosion. As result, the conditions of plants growth are improved (Kislenko, 1998).

The lignin contains no bonds that can be cleaved hydrolytically under conditions prevalent in cells. The strategy for its degradation, therefore, is based on free radicals reactions which are catalyzed by extracellular enzymes. The transformation of polyphenolic substances and lignins to humic acids at soil level is possible at least by two pathways: the abiotic oxidation catalyzed by minerals or metal salts; and the enzymatic oxidation by laccase and other oxidative enzymes. The abiotic polymerization of phenolic compounds has been shown by demonstration of the catalytic effects of certain oxides of Mn, Fe, Al,


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and Si to polymerize phenols to polymers. Also, the phenol oxidases (i. e. peroxidases or polyphenoloxidases – laccases) have an important function in the structurally assembly of the humic acid complex. The origin of the aromates may either be the aromatic moiety of the litter, root organic substances, or metabolites from litter decomposing microorganisms. From the chemical background of this reaction it is obvious that a free phenolic group is needed for this polymerization, a fact which is very important in the case of bioremediation of the area polluted with aromatic/phenol compounds.

Lignin and its derivatives are non-toxic by-products of the pulp and paper industry and are well known for their ability to bind heavy metal ions. With respect to fertilizers, lignins are capable of keeping micronutrients such as Cu, Co, Mn, and Zn in an available form under pH conditions that might normally cause insolubilization. Since the mentioned ions are more weakly bound than those made with other complexing agents, such as EDTA, they are more readily available to the plants.

The modified kraft lignin (KL) was also used to mimic the chemical structures of humic substances and evaluating them as new soil conditioning agents. A commercial softwood kraft lignin was modified by oxidation and sulfonation and the interaction between the chemically modified lignins and Al ions was studied. The obtained data indicate that the modified lignin not only can remove the toxic effect of Al, but it can also have a positive effect on the root elongation (Fig. 7). The radish growth test was conducted on culture solution at an exact pH and Al concentration.

Fig. 7. Effect of oxidated lignin (oxidated lignin – OL 8 875 ppm) on the growth recovery of Al treated (28. 8 ppm Al) radish roots with the initial pH of the soil being

adjusted by H2SO4 or KOH: ctr – control; Al- in the presence of Al ions; Al+OL 8 in the presence of Al ions and oxidated lignin (Katsumata and Gyosuke , 2002) It is interesting to note that the growth of the root was practically

prohibited in the absence of a lignin and in the presence of only 5 ppm of Al. However, with an addition of kraft lignin, root elongation slowly increased at the beginning and finally reached to the same level as that without Al at a concentration of ~ 350 ppm of KL. The kraft lignin itself had a positive effect on the growth of the radish root. When 70 ppm of KL was added to the culture


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solution, root elongation was about twice as much as that of the control conditions. A multitude of functional groups and a relatively high cation exchange of artificial humus substitutes also give them a great immobilization potential for noxious, inorganic and organic substances (complexation, salifiation etc.) (Fig. 8).

Fig. 8. Immobilization of toxic heavy metal Hg by the artificial humus

(Fischer and Schiene, 2002) Thus, the output of substances along with the seepage water can be

drastically reduced in the rehabilitation of problematic sites, if the artificial humus is used instead of the conventional mineral fertilizers.

3. 5. Composting

The degradation of organic matter during composting can be viewed as a

quick-motion film of the events leading to the mineralization of organic matter during humus formation and the humus cycle. During composting, both classic biochemical pathways of decomposition of organic molecules and the radical-mediated reactions are operating. Owing to the high organic matter content of the compost, the elevated temperatures during its operation, and the high C/N ratio at the beginning of composting (which leads to autolysis cycles with liberation of intracellular peroxidases), a milieu is created in which at least part of the radical reactions induced by white-rot fungi are also operating. Even lignin, when artificially added to the composting process is also degraded there (Hutterman and Majcherczyk, 1998). In these conditions along with the possibility to obtain the humus precursors, an alternative approach for bioremediation is the use of free radical-mediated reactions that are used by white-rot fungi and are in operation during composting, lignin biodegradation and in the humus dynamic of soils. Ligninolytic enzymes and mediators are active extracellularly, thus white-rot fungi may serve as better candidates for the bioremediation of highly apolar pollutants. If the conditions are not favorable for the spontaneous degradation of the pollutants, measures can be taken for the activation of the existing microflora. If the soil cannot be removed, the percolation with the oxygen and inorganic nutrient (nitrogen and phosphorus) or


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an electron acceptor (nitrate) can greatly accelerate the rate of toxicant degradation. If the soil can be taken from the site, the simplest method available for activation of the indigenous microflora is the process of landfarming. Here the soil is formed in mounds that are turned over at certain intervals. During this process, the soil is aerated and necessary substrates can be added. It must be considered, however, that with this treatment volatile compounds will be released into the air and any measured remediation may be only the result of transfer of this compound into the air.

Another approach, using organisms that grow on the contaminants, is the use of specially cultivated bacteria. Thus, pentaclorophenol (PCP) –contaminated soil was remediated with a bacterial strain (Pseudomonas) that mineralizes approximately 75 % of the PCP to CO2 , what means a relatively little residual toxicity in the soil as measured by seed germination and root elongation of some plant species and by toxicity to earthworms. These direct-toxicity measures showed the bioremediated soil to be indistinguishable from clean reference soil. There was, however, a change in lignocellulose based respiration. The change in ability of the soil to process lignocellulose may possibly reflect a change in community composition of the fungi, which are the predominant organisms involved in lignocellulose degradation. It was observed that plant-microbe symbioses may be sensitive indicators of soil ecological disruption. Rhizobium nodule formation and vesicular-arbuscular mycorrhizal (VAM) fungal infection were affected in the remediated soil even though several other plant related toxicity measures (germination and root elongation) were unaffected. Rhizobial and VAM fungi plant/microbe symbioses are ecologically important partnerships; particularly in natural or low agronomic input ecosystems where they represent respectively, the major means of nitrogen and phosphorous inputs to vegetation in terrestrial ecosystems.

The plant-microbes symbiosis may also be useful as indicators for the ecological effects and environmental safety of biotechnology products auch as bioremediating agents.

Suitable bacterial strains are isolated by a series of enrichment cultures, grown in a cheap food base, such as wheat bran, and added to contaminated soils. All bacterial processes have in common that the organisms use the xenobiotic substances as a substrate for the generation of energy and biomass production. A consortium of microorganisms involved in the soil formation, commercially available could be composed of:

1. Fungi: - Brown rot fungi – e. g. Trichoderma reesei, Trichoderma harzianum (fungi decompose plant material to glucose- carbon source) - White rot fungi – e. g. Phanerochaete chrysosporium (fungi decompose very quick the organic matter and are specific for lignin) - Mycorrizae fungi – soil may be improved with these fungi which prevent soil compactness, enable water to percolate in the soil, absorb water near the soil’s surface, stabilize soil and inhibit


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erosion. Mycorizae fungi help plants to acquire their nutrients 2. Bacteria:

- Athrobacter globuliformis, Streptomycetes baduiis (these bacteria live in close proximity with fungi and they utilize the decomposition by-products and convert them to carbon and nitrogen compounds)

- Nitrogen fixing bacteria: Azotobacter vinelandii, Rhizobium sp. (They use the gaseous nitrogen from the atmosphere and convert it to ammonia compounds. These bacteria produce growth-promoting hormones that increase root hair development, enabling the plants to accumulate a greater amount of nutrients).

- Pseudomonas fluorescens which produce antibiotics that attack and kill pathogen agents.

3. 6. Bioremediation

3. 6. 1. Phytoremediation There are two phytoremediation approaches that may be utilized in

tailings or other contaminated soils: a. phytostabilization or phytorestoration b. phytoextraction Phytostabilization involves metal inactivation as a result of the

establishment of vegetation with or without the use of chelating agents. This technique simply reduces the mobility of potentially toxic metals. Ideally vegetatitive species would be chosen which limit the uptake of metals by roots and subsequent translocation to the shoots. The lignin derivatives may be tailored to play a role in reducing metal uptake. Thus, by using ultrafiltration it is possible to separate lignins into different molecular mass fractions which could have selected properties that will enhance the uptake of micronutrients in nutrient deficient soils or decrease metal uptake where potentially toxic levels of metal exist.

Phytoextraction on the other hand attempts to increase the removal of metals from soils and generally through the use of hyperaccumulating plant species. Therefore, several plant physiological properties are important for phytoremediation, including potential metal accumulation, rate of metal translocation from roots to shoots, high biomass production, high tolerance to specific metals.

The addition of lignin derivatives to an acidic (pH 4.5) metal – contaminated feed solution could increase the quantity of shoot biomass. Also, the chlorophyll fluorescence measurements indicated that photosynthetic efficiency was improved with the addition of lignin derivatives.

Alternative natural substrates such as terpenes and flavonoids have been shown to enhance PCB (polychlorinated biphenyls) degradation to an equal or greater extent than biphenyl. The mechanisms by which natural plant products


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enhance degradation of PCBs are unclear. However, it may be likely that terpenes and lignin (and/or their metabolites) can induce PCB-degradative pathway. This assumption is based on the structural similarities between plant terpenes or lignin (and their metabolic products) and biphenyl (and its metabolites). The use of plant terpenes or lignin as inducer for PCB or PAH (polycilic aromatic hydrocarbons) degradative pathway has clear advantages: good availability, compatibility (nontoxicity) in natural environments and their ubiquity in the environment (Sung-Cheol et al., 2004). 3.6.2. Bioremediation by radical reactions An alternative approach for bioremediation is the use of free radical-mediated reactions that are used by white-rot fungi and are in operation during composting and in the humus dynamic of soils. The most efficient producers of extracellular enzymes that nonspecifically oxidize aromates by electron elimination known so far are white-rot fungi. They generate such a high redox potential, that this metabolic approach was termed “enzymatic combustion”, which means that they “burn down” all available aromates present in the proximity of the mycelia (Huttermann and Majcherczyk, 1998). The final fate of the compounds treated by this way depends on their chemical structure and presumably, on the enzymes that are involved. So far the following four different possible chemical pathways have to be considered: 1. Complete mineralization to CO2 and water: This is the optimal fate one would like to see when applying bioremediation. During the usual time frames by which bioremediation is applied, this only happens to part of the carbon atoms of the toxic compounds. 2. Incorporation into fungal or microbial biomass: After oxidation of aromatic compounds molecules are formed that are much more easily metabolized than the parental ones. These newly formed compounds can be funneled into the metabolism of the soil microflora and converted to microbial biomass. 3. Polymerization: Definite polymerization reactions occur during the degradation of lignin. Whether a certain compound is either degraded or polymerized by certain enzymes is dependent on the chemical structure of the very molecule. 4. Incorporation into the soil matrix: Another reaction that is usually neglected is the incorporation of the compound, or its transformation product, into the organic matrix of the soil. This pathway is not surprising, considering that all reactions catalyzed by lignolytic enzymes of the white-rot fungi go through radical intermediates, which are very reactive. Therefore, at least part of the reactions takes place with organic matter of the matrix leading to a fixation in the form of an insoluble and not readily accessible part of the humus structure.


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4. Conclusions Soil microorganisms degrade the lignins and their derivatives by

cleaving off units consisting of one, two or at most three phenolic acid moieties. These transformations have as result humus formation and they are accelerated when the modified lignins are used, especially in the case of those with a high degree of degradation. On the other hand, some chemicals from plants may be released from living leaves as volatiles or leachates, or from roots through exudation or sloughing off dead tissues. They also may leached from litter on the soil surface. Most of them are of polyphenolic nature. These chemical compounds influence the biological processes in a complex manner both at the soil level (in the humus formation) and plant development (by their intervention in the hormonal chain). Thus, in the last case it was possible to identify products with inhibitory (e. g. herbicides or pesticides) or with stimulatory action on different stages of plant propagation (seeds germination, plant grafting, plant development a. s. o. ). Also among them, the compounds that can act on the expression of heredity program in another plant were identified. The knowledge of these processes allows using the lignins and polyphenols in different ways to perform crops cultivation and bioremediation. That is why, the following aspects could be considered of interest in the future investigations: 1. a) participation of lignins in the humus formation (native lignins, separated and modified lignins), (bio)remediation, phytoremediation, a. s. o. b) utilization of lignins as slow release agents for pesticides, insecticides and herbicides and as soil conditioning fertilizers and sequestering agents c) investigation of synergic interaction between lignin, microorganisms and plants 2. The role of lignin and its derivatives in activating vital processes in plants (hormones, receptors, transducing molecules, transcription factors) with the following targets: flowering control, plant tissue culture, better growth rates, improved tolerance and possible application in: crop yield, seed, energy, biomass, pharmaceutical, higher sugar grasses, methane reduction, crop rotation time, diseases resistance, cold and drought. 3. The participation of lignins and their degradation products in the plant metabolism. 4. The influence of lignins and their degradation products on soil microflora and the effect of lignins on the structure of the soil. The modification of lignins by composting, with or without utilization of selected microorganisms having in mind the possibility to involve these processes in the increase of soil fertility or in bioremediation. 5. The use of lignins, their derivatives and polyphenols in the complex processes of bioremdiation. This review is based on our participation in the thematic network EUROLIGNIN (Coordination network for lignin in OE standardization, production and applications adapted to market requirements)-G1RT-CT-2002-05088.


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