Chemical characterization of the lignins of corn and soybean agricultural residues

Caracterização química das ligninas dos resíduos agrícolas de milho e de soja

Abstracts

Corn (CR) and soybean (SR) culture wastes were submitted to extraction with organic solvents for lignin isolation (LGS). The obtained lignin was chemically characterized, and based on studies of functional groups and microanalyses, it was possible to determine the minimum formula. LGS of CR has characteristics that resemble wood and of bamboo lignin, possessing a larger amount of methoxyl groups and vanillin.

Lignin; culture residue; chemical characterization; corn residue; soybean residue


Foram utilizados dois restos de cultura, resíduo de milho (CR) e resíduo da cultura de soja (SR) e o material foi submetido à extração com solventes orgânicos para isolamento da lignina (LGS). A lignina assim obtida foi caracterizada quimicamente. Com base em estudos de grupos funcionais e microanálise foi possível determinar a fórmula mínima para as ligninas. A LGS de CR tem características que a assemelham à lignina de madeira e do bambu, apresentando maior quantidade de grupos metoxila e de vanilina derivada.

Lignina; resíduo de cultura; caracterização química; palha de milho; palha de soja


Chemical characterization of the lignins of corn and soybean agricultural residues

[Caracterização química das ligninas dos resíduos agrícolas de milho e de soja]

E.O.S. Saliba1, N.M. Rodriguez1, D. Piló-Veloso2, S.A.L. Morais3

1Departamento de Zootecnia da Escola de Veterinária da UFMG

Caixa Postal 567 - 30123-970 - Belo Horizonte, MG

2 Departamento de Química - ICEx - UFMG.

3 Departamento de Química - UFU

Recebido para publicação em 20 de janeiro de 2001.

Recebido para publicação, após modificações, em 16 de agosto de 2001.

E-mail: saliba@vet.ufmg.br

ABSTRACT

Corn (CR) and soybean (SR) culture wastes were submitted to extraction with organic solvents for lignin isolation (LGS). The obtained lignin was chemically characterized, and based on studies of functional groups and microanalyses, it was possible to determine the minimum formula. LGS of CR has characteristics that resemble wood and of bamboo lignin, possessing a larger amount of methoxyl groups and vanillin.

Keywords: Lignin, culture residue, chemical characterization, corn residue, soybean residue

RESUMO

Foram utilizados dois restos de cultura, resíduo de milho (CR) e resíduo da cultura de soja (SR) e o material foi submetido à extração com solventes orgânicos para isolamento da lignina (LGS). A lignina assim obtida foi caracterizada quimicamente. Com base em estudos de grupos funcionais e microanálise foi possível determinar a fórmula mínima para as ligninas. A LGS de CR tem características que a assemelham à lignina de madeira e do bambu, apresentando maior quantidade de grupos metoxila e de vanilina derivada.

Palavras-chave: Lignina, resíduo de cultura, caracterização química, palha de milho, palha de soja

INTRODUCTION

One of the greatest changes in human diet began in 1880, with the development of cereal processing techniques, and obtaining almost pure wheat flour along with the increment of sugar availability, that was only a pharmacological product previously. The industrialization of foods aimed to extract the noblest portions for human consumption or for industrial ends (Ruben, 1975, apud Bose & Machado Filho, 1984). Due to food industrialization and growing demand to feed mankind, other products were used such as waste residues, constituted mostly of structural carbohydrates, which are known as fiber, or according to Theander (1977), as a dietary fiber. Annual cultures such as corn and soybean occupy about 26 million hectares. Therefore, more than 200 million tons of culture residues, mainly corn and soybean, would be annually available (Anuário..., 1996). On the other hand, the culture wastes are available to be used for feeding ruminants exactly during the period of green forage shortage due to cold time or dry periods of the year.

Lopes & Nunes (1992), studying the potential use of Brazilian feed resources affirmed that about 40% of the estimated 130x108 cattle population in Brazil, is found in savannas (cerrado), area that includes more than 200 million hectares. Production levels per animal and area have been low and of lower quality for several decades due to low fertility of the soil and acidity, coupled with traditional burning of agricultural residues.

The potential of culture residues and by-products, particularly in savanna region is tremendous. Agricultural residues are poor in nutrients and available from a great variety of cultures of agricultural enterprise of Brazil. Their satisfactory use in animal feeding requires understanding of their nutritional limitations, processing, economic aspects and opportunity of use.

Research in the field of ruminal digestion has been shown clearly that straws can supply most of requirements for low productivity animals, and can be part of a diet fraction for animals high productivity (Mamorama et al., 1991; Sewalt et al., 1996). The problem for fibrous materials utilization is the lack of knowledge of its chemical constitution without which its action cannot be foreseen in animal organism (Chenoste & Reiniger, 1989).

Fiber, mostly from cellular wall, has cellulose as the main component. The complex structure of long cellulose chains form microfibrils that join in bunches constituting macrofibrils, which associated with several substances, most of what carbohydrates, form cellular wall. Most walls, especially of woody tissue, are impregnated with lignin (Ferri, 1978). Fibrous residues of cultures can become rich foods in energy for the cattle, if the holocellulose is free from lignin (Mamorama et al., 1991).

Straws of small grains possess, in general, low nutritious value. Wheat straw is probably one of the lowest quality although that of barley is slightly better. Oat straw seems to have a high nutritious value. For most of the straws, lignin is the main cause of low digestibility (Jackson, 1977).

Lignin analyses of ground straw (LGS) of CR and SR will be the main objective of this work, which aims to characterize its macromolecule. Structures of lignins will be discussed based on chemical evidences, such as results of elementary analysis, analysis of functional groups, degradation products through the oxidation reaction with nitrobenzene, and combinations of these methods with spectrometric analysis in infrared (IR), nuclear magnetic resonance (NMR) of 1Hand 13C.

MATERIALS AND METHODS

Two culture wastes residue of corn (CR) and residue of soybean (SR) were submitted to extraction with organic solvents for lignin isolation (Saliba et al., 1998).

Acetilation of LGS (LGSa): To 100mg of the sample, 10ml of a mixture of acetic anhydride: pyridine (1:1) was added. Soon after, the resulting solution is left to reflux at 50ºC for 24 hours, then diluted with water until completing precipitation. The resulting solution was filtered and the obtained precipitate sequentially washed with water, followed by a diluted solution of hydrochloric acid and again with water. Finally, the sample was dried in a stove at 70ºC until a constant weight was obtained.

Determination of methoxilyc groups (OCH3): About 2 to 6mg of the sample (LGS) were measured in reaction balloon. For this sample 0.1g of phenol, 1.2g of potassium iodine and 2ml of orthophosphoric acid were added to avoid exhaust gas. A condenser was coupled to the balloon, a tube containing ascarite and exit tube for gas, connected to a flask containing acetic acid, bromine and sodium acetate solution for absorption.

The absorption solution was composed of 200ml of acetic acid, 1ml of bromine and 20g of sodium acetate. As it became warm, reaction was maintained for one hour at 200ºC in an inert atmosphere, produced by a flow of nitrogen, dragging the formed methyl iodine. After this and removed from heating, the content of absorption flasks was transferred to an erlenmeyer (150ml), containing 10ml of 20% sodium acetate solution. The solutions of absorption flasks were mixed with the main solution.

To remove the excess of bromine of absorption solution, 2ml of 2% formic acid was added, under agitation, and the system was closed and left to rest for five minutes. Soon after, 5ml of 10% sulfuric acid and 0.1g of potassium iodine was added, it was mixed and left resting for 10 minutes, in darkness.

The liberated iodine was titrated with 0.02N sodium thiosulphate. Almost at the end of the titration, when the solution was light yellow, 2ml of 0.5% starch solution was added as indicator. A test in white was accomplished in the same conditions. The method was performed as described by Klimova (1977).

Determination of total hydroxyls for conductivity (OH): Lignin samples were diluted in 30ml of acetone:ethanol:water mixture (5:10:15). This solution was placed in a balloon of three mouths, in one of which the electrode of the instrument used to measure conductivity was introduced. The balloon was still connected to a vessel for titration and a driver of nitrogen flow, to maintain the atmosphere inert.

The initial value indicated in the instrument was registered and, after of each drop of lithium hydroxide, the value of respective conductance was registered. Before each reading, the solution was properly mixed and allowed to stabilize. The addition was proceeded with at least 3ml of 0.1N LiOH. A titration curve was plotted and the value of equivalence point used to calculate the test of total hydroxyls (phenolics + carboxylic) (0.091N lithium hydroxide) was included.

Determination of total carbonils (CO): In a 50ml balloon containing 0.2 hydroxylamine and 15ml pyridine 0.2g of LGS were added. The solution was maintained to reflux at 95ºC for 24 hours. After cooling, 15ml of water was added, forming a precipitate. The solution was filtered and the precipitate washed with 15ml of 10% HCl and soon after with water even neutral pH. Finally, it was left in a dessicator for three days, after which it was dried in a stove at 50ºC up to constant weight.

The conditions for obtaining the NMR spectrum of LGS 1H and 13C, were, respectively: Bruker DRX 400 (spectrometer of nuclear magnetic resonance); DMSO-d6 and CDCl3 solvents; scanning number of 30,000 and 1,000 and 0.5 as time of relaxation.

Nitrobenzene oxidation: In a reactor Parr model 4841 of 10ml, 0.025g of LGS, 9ml of 2.5N NaOH and 1ml of nitrobenzene were added. Soon after, the system was closed leaving reaction to occur at 170ºC for three hours. The mixture was transferred oxidized to continuous extraction with chloroform for three hours, to eliminate the nitrobenzene excess. Soon after, the aqueous solution was acidified with 3.0N HCl for further continuous extraction with chloroform, for 20 hours. Chloroform extract was concentrated under reduced pressure and transferred for a 5ml reaction flask. Thereafter, 4mg benzofenone were added (internal pattern) and 1ml of BSTFA (agent sililante) to concentrated extract. The flask was closed and maintained one hour at 70ºC to process the reaction. For determination of lignin degradation products, the solution, after cooled, was submitted to chromatography (gas - liquid), in Varian chromatography 3400 with Shimadzu integrator, and the following conditions: capillary column SE54 - 30m; gas flow (2ml/min); detection temperature 270ºC; injector temperature 270ºC; column temperature 120ºC for 1 min, 10ºC/min up to 250ºC; flames ionization detector (FID); injected volume - reason split 1/70.

Determination of moisture in lignin: It was analyzed the difference of weight of the initial lignin and the lignin after drying for six hours in stove (Biomatic 1305) at 105ºC.

Determination of lignin ash: An oven was used (Fornos Lavoisier) at 600ºC, during 1h, to determinate ash in lignin.

Determination of inorganic components: Lignin ashes were evaluated for inorganic components using X-RAY fluorescence spectrum, obtained in a Rigaku model Geigerflex.

Infrared spectroscopy (IR): The spectrometer was used in IR, model I Watson Galaxy series FTIR 3000, in pellets of KBr after 20 minutes homogenization.

RESULTS AND DISCUSSION

Table 1 presents some data of the chemical composition of LGS of CR and SR. The yield of LGS of SR was 3.52% and of LGS of CR was 5.77%.

The results consisted of:

A determination by X-RAY fluorescence of lignin ashes was made and the results of inorganic components are listed in Table 2.

The number of inorganic elements present in LGS of CR and RS was inferior to that observed for lignin of ground wood (LGW) of the Eucalyptus grandis studied by Morais (1987), where presence of Ca was verified in addition to S, Mg, Fe, Ti, K, Si, F, Cu, Ba, Al and Na.

The Si is considered to be originated from the silica present in sample, that was of the order of 0.72% in CR. The presence of Cl can have origin in the agricultural defensive commonly used in cultures. Presence of Ti in ashes of LGS of CR and not in LGS of SR, can be explained by the different origin of culture residues. On the other hand, it can also be due to the cutter machine, that was different in each farm.

Fengel & Wegener (1984) described the main inorganic components of wood ashes as being K, Ca and Mg (in case of tropical wood Si was also observed).

Some problems are related to the technique of lignin determination. Giger (1985), in extensive revision on methods of lignin dosage, concluded that this is a substance of difficult dosage, because most of the techniques are based on the ability of sulfuric acid in dissolving lignin. Once it is not totally soluble, dosage is harmed. The most common techniques involve the determination of Klason lignin (KL) are H2SO4 and detergents, and those less frequently used employ enzymes (amylases and proteases). However, comparisons among methods of determination of lignin content are scarce.

According to Sarkanen & Ludwig (1971), in case of grasses, the procedure for determination of KL should be modified, mainly because of ash content and condensed protein, or both. It should be carefully verified the presence of protein linked to lignin because it can increase KL weight. The range of error due to protein presence in KL is appreciable. Another problem described in the literature is linked to the use of the factor 6.25 [The constant 6.25, correction factor used in the calculation of the gross protein (CP), is based on the medium text of nitrogen (N) contained in the proteins in a general way (16%). Dividing 100 by 16 equals to 6.25, a factor of conversion of N considered for CP (Silva, 1981)] toestimate protein linked to lignin as this factor can be good for pure proteins, but composition of protein fragments linked to lignin may not be the same of the original protein.

Another observation with KL recovery is that a partial hydrolysis may happen in 72% H2SO4 of ester groups of those derived from p-cumaric and ferulic acids. Giger (1985) showed that in herbaceous plants, some lignin was dissolved during determination of KL.

Reeves III (1993), studying the technique of KL determination in wheat straw, obtained similar results to those of Sarkanen & Ludwig (1971), with values of 0.56% nitrogen.

In the present study a comparison was made between the N contents of KL, obtained from CR and SR, with those obtained from LGS of CR and SR. Data are shown on Table 3.

For the data in Table 3 it can be verified that KL was contaminated with nitrogen and LGS of CR and SR presented the same test of N being inferior to the respective KL, checking that are purer lignins and therefore own for the characterization study.

In a study of lignins of Eucalyptus grandis, Morais (1987) verified that the way of obtaining lignin interfere in the data of its elementary analysis. Thus, for this vegetable LK presented a %C = 63.22; %H = 4.28; %O = 32.24 and %N = lines, while native lignin (NL) that is obtained by extraction with organic solvents, revealed a %C = 56.50; %H = 6.40. %O = 37.76 and %N = 0.34.

Fengel & Wegener (1984) showed values for elementary analysis of lignins from soft wood varying between %C = 60 - 65% and in hard wood %C=56–60%. It also showed that these data are in agreement with the high content of oxygen in hard wood.

Chen (1991) proposes the elementary composition of LGW of bamboo as being %C = 61.66; %H = 5.53, and %O = 32.81.

Analysis of functional groups in ground straw lignins: Groups OCH3 determination is important for lignins characterization as it is known that lignins contain less than 1 mol of OCH3 per phenylpropanoid (C9) unit they are of the type guayacil (G), that is characteristic of soft wood. Lignins that possess more than a mol of OCH3 groups per C9 unit is of the type guayacil-syringil (G-S), being characteristic of hard wood and grasses (Morais, 1992).

The methoxyl group was determined in the present work by technique developed by Vieböck and modified by Klimova (1977). The method is based on methoxyl decomposition with concentrated hydrochloric acid, involving formation of alkyl iodine. Alkyl iodine is absorbed in solution containing bromine and acetic acid to form bromine iodine and alkyl bromide, in presence of acetate.

Involved reactions were:

The volumetric method of Vieböck is used for indirectly methoxyl determination by analysis of methyl iodine, obtained after reaction of the sample with HI. Thus, to measure methyl iodine being formed, it is absorbed in a solution of acetic acid containing sodium acetate and bromine, according to the following reaction:

At the end of the reaction, bromine excess is destroyed by addition of aqueous solution of 2% formic acid. It is proceeded by the addition of H2SO4 + KI to generate the iodic acid. This, reacting with the iodate liberates iodine, which will be determined by titration with sodium thiosulphate, using starch as indicator.

Therefore:

Vs=volume of thiosulphate of waste sodium after titration of the sample ,

Vb=volume of thiosulphate of waste sodium after titration of the white,

f=correction factor for standardization of the sodium thiosulphate solution,

N=normality of the solution of sodium thiosulphate,

m =amount of sample, in mg.

The basic alteration introduced in this method by Klimova, in 1977, was the use of a mixture of sulfuric acid and potassium iodine, providing the production of iodic acid directly in the reaction flask.

Table 5 shows the data of percentages of methoxyl (OCH3) of lignin from ground straw of corn and soy culture residues (LGS of CR and SR).

Data in Table 5 show methoxyl content relatively smaller for LGS-SR.

The content of OCH3 found in lignin of ground wood (LGW) of Eucalyptus grandis by Morais (1992), was 22%. In a work from 1987, Morais found native lignin (NL) of Eucalyptus grandis to be 5.86%. It can be seen that this lignin (NL) resembles each other more with LGS-CR in this work.

Fengel & Wegener (1984) found that hard wood possess a methoxyl content varying from 18 to 22% and soft wood present the variation between 12 and 16%. Chen (1991) found 19.42% of methoxyl for LGW of the bamboo. Harkin (1973) affirmed that methoxyl content in grasses is variable, in spite of containing mainly p-cumaril units, it can vary from 6.4 to 19.3% in different species.

The data in Table 5 indicate that LGS-CR and LGS-SR, for presenting low methoxyl contents, are derived from p-hydroxycinnamyl and/or coniferyl alcohol, with predominance of the first.

Determination of total phenolic hydroxyls: Considering the precursory aromatic substructures of lignin, it is verified that each one possesses a phenolic and aliphatic hydroxyl (OH). Lignin studies indicate that phenolic OH is present in only 20 to 40% of these monomers, demonstrating that most was sterified during the polymerization process as it can be seen below in the reaction for lignin formation (Morais, 1992), where the process of radical oxidation of condensation is schematized.

The study of phenolic hydroxyl contents of lignin provides information on its condensation and/or on the nature of C6C3 units (Morais et al., 1994).

Several methods can be applied for determination of phenolic hydroxyls. In this work, conductivity titration was used (Morais, 1987). The reactions involved are simple neutralization of phenolic hydroxyls by lithium hydroxide. The percentage of total phenolic hydroxyls was determined directly by the equation:

N = normality of the solution of used hydroxide

V = hydroxide volume in, ml used for titration

m = sample mass, in mg

The total content of phenolic hydroxyls was expressed as a function of its molar amount by minimum formula, in the equation:

N, V, m = same of the equation (1)

MW/C9 = molecular weight of lignin/C9

The data of MW/C9 of this healthy work:

Table 6 shows results obtained from samples in the study considering, in case of the total phenolic hydroxyls /unity C9, respective molecular weights MW/C9 previously mentioned.

Literature mentions that a mol of OH/C9 in the wood would be from 0.2 to 0.4 and in the protolignin of 0.1 mol OH/C9. A higher percentage of it can be due to the ground process. Morais (1992), in a work of characterization of LGW of the E. grandis, found 2.3% of OH total phenolic and 0.28% of OH/C9.

In several types of lignin of E. grandis, the following percentages of OH total phenolic were found: technical lignin (TL) = 5.48, KL = 6.62 and NL = 7.40; variations are due to the conditions of obtaining lignins (Morais, 1987).

Chen (1991) found the value of 1.49% for OH total phenolic of LGW of bamboo. Comparatively, data in Table 6 meet closer of those of TL and LN of the E. grandis.

Determination of carbonyl groups: Carbonyl groups (CO) are other important functional groups in lignins characterization. Several methods can be used to determinate total CO content. In the present work, reaction and determination of N percentage added after this reaction was used. Hydroxylamine reacts with all types of carbonyl present in the macromolecule of lignin. The calculation of that percentage is made applying the equation:

where:

N1 = percentage of nitrogen after reaction

N = percentage of nitrogen before reaction

When CO percentage is known then, its molar content can be determined by methoxyl mol (CO/OCH3), using equation (4) and, to calculate C9(CO/C9) molar proportion.

Table 7 presents CO percentages, as well as CO/OCH3 ratio for LGS of CR and SR.

For LGW of E. grandis, Morais (1992) found values of %CO = 2.54 and CO/OCH3 = 0.13. For TL, KL and NL, the same author observed values of 2.20, 1.49 and 2.68, respectively, for CO percentage (Morais, 1987).

Studying CO groups in lignins, Marton & Adler, apud Morais (1992), found in Bjorkman lignin (BL) relationship of 0.2 (CO/OCH3).

Lignins are formed from C6C3basic units, as previously mentioned. Therefore, previously knowing percentages of carbon (%C), hydrogen (%H) and methoxylics groups (% OCH3), it is possible to establish a minimum formula, based on its phenylpropanoid unit C6C3 (C9). The minimum formula of lignins are represented by the general equation

Values of b and d are based on percentages of the elements used in the equations:

Table 8 presents the minimum formulas calculated for LGS of CR and SR.

Morais (1987, 1992) found MF and MW several lignins of E. grandis that are listed in Table 9. Molecular weights are of the order of that observed in LGS of CR, being MW of LGS of SR slightly smaller.

Fengel & Wegener (1984) found MF of the lignin of soft wood typically as C9H9,1O2 (OCH3)0,92. According to Freundeberg & Nush (1968), to be considered pure lignin, its minimum formula should correspond to C9Hg O2(H2O)h (OCH3)i, where g= 10 - i - 2, i should be between 0 to 1.5, and h should not be larger than 1. Therefore LGS of CR and SR fill the necessary requirements to be considered genuine lignins that is to say, pure lignins.

As a method that preserves aromatic rings (Fengel, 1984), the reaction of lignins oxidation with nitrobenzene allows degradation of the macromolecule aiding to the study of structure. It is worth to remind that the oxidation of soft wood produces vanillin as the main product. Hard wood form vanillin and syringaldehyde, while grasses, besides these two aldehydes, produces p-hydroxybenzaldehyde.

In Table 10, giving sequence to the studies of characterization of LGS of CR and SR, results obtained through oxidation reactions with nitrobenzene are presented. The formed products were silylation and analyzed by gas-liquid chromatography (GLC).

The times of retention found were: H=4.33min; V=6.13min; AH=7.11min; S=8.03min.

Although just traces of degradation products have been detected, LGS of CR and SR demonstrated the same characteristics of grass and wood lasts lignins presenting as oxidation product with nitrobenzene a mixture of H, V and S. LGS of CR presented a smaller amount of ah that LGS of SR. LGS of CR already presented an elevated content of v that is in agreement with its largest methoxyl text when compared with LGS of SR. The presence of little amount of these oxidation products in LGS, is in agreement with its low content of OCH3 in relationship LGW of the E. grandis.

Lignin of grass crop residues presented a larger vanillin concentration in relation to that of leguminous. It is known that vanillin is derived from ferulic acid (unit guayacil). Grass and leguminous lignins have been poorly studied. Lignins of forages present variable compositions of the extract obtained from oxidation with nitrobenzene, indicating differences in its composition, that can affect its nutritional value. For example, syringaldehyde obtained by lignin oxidation with nitrobenzene was more negatively correlated with the digestibility of cell wall in grasses than in leguminous (Buxton & Russell, 1988). Like this, while the high lignin content is generally associated with low digestibility, its chemical nature is important, because it defines the largest or smaller inhibition of digestibility in different types of plants.

It is known that oxidative degradation of lignin results in liberation of aldehydes, ketones and carboxylic acid, which help to define its structural type. According to Faix et al. (1985) apud Akin (1989), different lignin types vary in degradation by microorganisms, and the lignin rich in syringil is degraded more easily than those in guayacil.

Concentration of carbonyl group and molar value of these groups in lignin depend on type and nature of each vegetable species (Chen, 1991).

LGS of CR and of SR were shown as lignin H-G-S, that occur in grass tissues (Gramineae; monocotyledonous angiospermae). Those are usually composed of core type G-S lignins and groups containing acid type 4-hydroxycinamic (AH) (Chen, 1988).

For the data in Table 6 and in studies of CG, respectively, in LGS of CR and of SR, abundance of free OH, in larger proportion that LGW of E. grandis (wood lasts), and presence of units H or AH and of G or S with free OH were observed. Characteristic of these lignins can favor formation of the complex lignin-carbohydrates.

CONCLUSIONS

Results obtained in this work and considering a parallel work with the composition of wood lignins it can be observed that lignin of corn straw has characteristics that resemble wood and bamboo lignins, with larger content of O and smaller of C in its minimum formula, smaller relationship carbonyl/methoxyl, larger amount of methoxyl groups and guayacil (vanillin). It is still not known, if these groups have some toxic effect on the ruminal bacteria, but it seems reasonable to infer that, being a more rigid lignin, animals will have a greater difficulty to break into fragments during rumination, with two clear consequences, larger physical barrier for bacteria to reach digestible tissues of the vegetable (effect Hottel) and a larger time of permanence in rumen, that causes decrease of voluntary intake of feed. It can be expected, that the digestibility and the voluntary intake of corn straw is inferior to that of soybean straw.

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Publication Dates

  • Publication in this collection
    22 July 2002
  • Date of issue
    Feb 2002

History

  • Accepted
    16 Aug 2001
  • Received
    20 Jan 2001
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