Chemical and infra-red spectroscopic study of humic and fulvic acids from composted poplar bark

Biological Wastes 19 [1987) 205-214

Chemical and Infra-red Spectroscopic Study of Humic and Fnlvic Acids from Composted Poplar Bark Ezio Roletto* & Maria Paola Luda+ * Department of Analytical Chemistry and tInstitute of Macromolecular Chemistry, University of Turin, via Pietro Giuria 5, I 10125, Italy (Received 24 October 1985; revised version accepted 6 June 1986)

A BS TRA CT Humic (HA) and fulvic (FA ) acids were extracted from poplar bark after 12 and 30 months of composting and characterized through functional groups analysis and FT-IR spectra. The extraction and purification procedures were those recommended by the International Humic Substances Society ( IHSS). Total acidity and COO H, total OH and C = O contents of FA were always greater than those of the corresponding HA. Carboxyl groups were the major components of total acidity. Phenolic OH groups content of HA was always higher than that of the corresponding FA, whilst the alcoholic OH groups content was higher in FA than in HA. There were no significant differences between HA.extracted after 12 and 30 months of composting, but some differences were shown by the corresponding FA.

INTRODUCTION In previous investigations, the composting o f poplar bark (Roletto et al., 1985), either alone or mixed with residues from the agrochemical industry, was studied. Attention was focused upon the influence of the duration of composting and the addition of organic wastes on the amount and quality o f the humic materials. Since our research project aims at investigating interaction between these humic materials and metal ions b o u n d into the structures of soil minerals, we have extended our studies to the analytical characteristics o f humic (HA) and fulvic (FA) acids. In this paper, data are reported concerning the elemental composition and functional groups 2o5 Biological Wastes0269-7483/87/$03.50 © Elsevier Applied Science Publishers Ltd, England, 1987. Printed in Great Britain

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analysis of HA and FA extracted from two manures obtained after 12 and 30 months composting of poplar bark. Spectroscopic information obtained by infrared Fourier transform spectroscopy is also furnished. The results of this investigation are compared with those found in the literature and refer to HA and FA extracted from different soils and from decomposing plant materials.

METHODS Separation of humic and fulvic acids HA and FA were extracted from composted poplar bark after 12 and 30 months of microaerobic composting. Some analytical characteristics of the composted materials and the extracted humic substances can be found in a previous work (Roletto e t al., 1985). In the present study, the extraction and purification procedure of HA and FA was according to the International Humic Substances Society (IHSS). Briefly, the method was as follows: the humified poplar bark was air-dried, ground to pass a 60 mesh sieve and then dried at 105°C to constant weight. The material was suspended in a 1M solution of HCI (sample to solvent ratio: 1/10, w/v), the pH being initially adjusted to a value between 1 and 2. The suspension was stirred for 1 h, left standing for at least 12 h and then centrifuged; the supernatant (called SF1) was given the XAD-8 resin treatment of FA. The residue was neutralized with 1M NaOH to pH 7.0; a solution of0"lM NaOH was then added to a final sample to solution ratio of 1/10 (w/v) and the suspension shaken intermittently under nitrogen at room temperature for 6h. After centrifugation, the supernatant was acidified with 6M HCI to pH < i'0 and allowed to stand at room temperature for at least 12 h under a blanket of nitrogen. The coagulate (HA) was separated by centrifugation from the supernatant (called SF2) which was given the XAD-8 resin treatment of FA. Purification of humic acid The HA fraction was dissolved in a minimum volume of 0"IM KOH under nitrogen and KCI was added to make the system 0"3M in ionic strength. After allowing the solution to stand for 6h, suspended solids were removed by centrifugation. Humic acids were then reprecipitated with 6M HC1 solution. The coagulate was separated by centrifugation and shaken for 24h at room temperature with a 0-1M HCI:0"3M HF solution until the ash content remained constant: this was usually achieved after the third

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treatment. The suspension was then filtered through a 0-45 ltm membrane (Gelman, GN-6) and the residue washed with twice-distilled water to a negative C1- test with AgNOa. Following this, the humic acid fraction was freeze-dried ( - 5 Y C ; 60-100mtor) and stored under nitrogen at - 1 8 ° C .

Purification of fulvic acid The SF1 fraction was adsorbed on a column of XAD-8 resin (Amberlite, R o h m & Haas, 20-50 mesh) which had been pre-treated according to T h u r m a n et al. (1978). The bed surface/bed height ratio of the resin was 1/15cm; the eluent flow rate was 10 bed volumes per hour and the bed volume/weight of the initial sample ratio was 0-5 cm 3 g - t. The column was rinsed with 0.65-0.70 bed volumes of bidistilled water and the fulvic acids fraction eluted with 1 bed volume of N a O H 0.1M and 3 bed volumes of bidistilled water. The eiuate was brought to pH ~ 1 with 6M HCI and HF was added to a final concentration of 0-3M HF. The same procedure was repeated with the SF2 fraction (bed volume/weight of initial sample ratio of 1 cm 3 g - 1). The eluates f r o m SF1 and SF2 treatments were combined and passed through XAD-8 resin in a plastic column with a bed volume/weight (initial sample) ratio of 1 cm a g-1. After elution of the fulvic acid fraction with 0"IM N a O H (1 bed volume) and bidistilled water (2-3 bed volumes) the eluate was passed over a column of cation-exchange resin (BIO-RAD, AGMP 50) in the H-form, for conversion to fully protonated form. Finally, the fulvic acids fraction was freeze-dried and stored under nitrogen.

Chemical analysis Methods for evaluating ash content and pH value, as well as performing elemental analysis and gel-permeation chromatography, have been described elsewhere (Roletto et al., 1982; Roletto & Ottino, 1984). Total acidity, carboxyl groups, total hydroxyls and carbonyls were determined according to the methods reported by Schnitzer & Kahn (1972). The amounts of phenolic hydroxyls and alcoholic hydroxyls were estimated indirectly. Infrared spectroscopy The infrared analysis was carried out in duplicate with a Perkin-Elmer F T I R 1500 spectrophotometer, using the KBr pellet technique. To prevent adsorption of hygroscopic moisture, all samples and KBr were stored for at least 48 h in a desiccator above P205 (Granusic, Baker Analyzed Reagent)

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and the KBr-humic material pellet was allowed to stand overnight in the same desiccator before scanning. Spectra were recorded with 4 c m - t resolution and trapezoidal apodization and normalized by means of the ABEX program. This program cannot be used when the strongest band has an absorbance value higher than 1.5. This was taken into account in the sample preparation of KBr pellets.

RESULTS AND DISCUSSION

Elemental and functional groups analysis Elemental compositions of HA and FA are given in Table 1; data from functional groups analysis are reported in Table 2. Data on 'model' soil FA and HA (Schnitzer, 1978) are reported in Table 3. The values of HA and FA extracted from composted poplar bark agree with those from previous works on soil humic materials, indicating that: humic acids generally have higher C and N, but lower O, content than the corresponding fulvic acids; fulvic acids show total acidity and C O O H groups content higher than the corresponding humic acids; total acidity is related almost entirely to the carboxyl content of HA and FA, and OH phenolic groups are not major contributors to total acidities (Hatcher et al., 1981). We are aware that the current analytical methods for oxygen-containing functional groups, as has TABLE 1 Elemental Analysis o f H u m i c a n d Fulvic Acid F r a c t i o n s

Variable

HAl2

HA30

FA I 2

FA30

Ash a NID Cb NID Hb NID Nb NID O + Sc

395 + 0'42 3 51.02+0-44 3 4-37+0-06 3 2.05 + 0-04 3 42.56 24-9 117

183 + 0-52 3 51-52+0.30 3 4.36+0-17 3 2-44 _ 0.01 3 41.64 21.1 118

1'10 + 0'20 3 49-89+0.87 3 3.31 + 0 . 7 0 3 0.85 + 0-05 3 45.95 58.7 151

400 _ 040 3 42.31 + 0 . 8 5 3 3.29+0.16 3 1.38 _ 0.18 3 53.02 30-7 12"9

C/N C/H

a Weight %, moisture-free basis. b Weight %, moisture- a n d ash-free basis. c Calculated by difference. N I D , N u m b e r o f i n d e p e n d e n t determinations.

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TABLE 2 Distribution of Oxygen-containing Functional Groups (mew'g) of HA and FA (All data are on a moisture- and ash-free basis) Functional group

HAl2

Total acidity NID COOH N|D Total OH NID Phenolic OH Alcoholic OH Total C = O NID

6.5+0.44 3 4.3 + 0.20 4 7.94-0.13 3 2-2 5.7 2.8 + 0"35 3

HA30

6-$-+-0-16 3 4-5 Z 0-34 4 4-$'_0-75 3 2.3 2.5 3-4 4- 0-92 3

FAI2

FA30

7.9+0.90 3 7.4 + 0.32 4 9.6+0-10 3 0-5 9"1 5"8 4- 0.67 3

9-3+0-68 3 8.2 + 0.28 4 10.74-0-80 3 1-1 9-6 3-5 4- 0-35 3

NID, Number of independent determinations. a l r e a d y b e e n p o i n t e d o u t ( S t e v e n s o n & G o h , 1972), are s o m e w h a t n o n specific a n d c a n lead to e r r o r s in their q u a n t i t a t i v e e s t i m a t i o n . N e v e r t h e l e s s , we t h i n k t h a t t h e y c a n be useful in a c o m p a r a t i v e s t u d y s u c h as the p r e s e n t one. T h e i n f l u e n c e o f the h u m i f i c a t i o n p e r i o d u p o n the c h e m i c a l c h a r a c t e r i s t i c s o f H A a n d F A is quite different. T h e d a t a in T a b l e 1 a n d m o s t o f the d a t a in T a b l e 2 s h o w no significant differences b e t w e e n H A 1 2 TABLE 3 Mean Values of Elemental and Functional Groups Analysis of 'Model' Soil Humic and Fulvic Acids (Schnitzer, 1978) Element ( % )

C H N O+ S Functional groups (meq/g )

Total acidity COOH Phenolic OH Alcoholic OH Total C ~ O

HA

FA

56-2 4.7 3-2 36"3

45.7 5"4 2"1 46.7

HA

FA

6.7 3'6 3"9 2-6 2-9

10"3 8"2 3.0 6"1 2.7

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Ezio Roletto, Maria Paola Luda

and HA30; on the other hand, there are marked differences between the characteristics of FA extracted from composted poplar bark at different stages of the humification process. In fact, FA12 shows a higher C content and a lower N and O content than FA30; moreover, FA30 has a higher value of total acidity, COOH and OH phenolic groups content than FA12. All these data could be easily interpreted by assuming that FA represent initial products of humification and further progress of this process results in the formation of humic acids. Moreover, the evolution of the C/3'4 ratio from higher (58.7 for FA12) to lower values (21.1 for HA30) is usually linked to the degree of stability of humic materials. On the other hand, the reduction in the C/H ratio, which is generally considered to be parallel to the degree of condensation or aromaticity of the humic fractions, could suggest a decrease in the condensation of aromatic nuclei as humification increases. This finding seems to be consistent with recent views (Hatcher et al., 1981) on the humification of organic remains in both terrestrial and aquatic environments, suggesting that paraffinic structures from microbial exudates can play a conspicuous structural role in the formation of HA.

Infrared spectra The Fourier Transform infrared spectra of the HA are given in Fig. 1; those of the FA are shown in Fig. 2. The former exhibit bands near 3400 c m - t, 2930, 1720, 1630 and 1200cm-1; less strong absorption bands are present at 2600, 1510, 1450, 1420, 1120 and 1030cm -~. Fulvic acid spectra show bands near 3400, 1720, 1630, 1200 and 740 c m - i; less important absorption bands occur at 2930, 2600, 1510, 1120, 1080 and 1060cm-~. IR spectra of humic and fulvic acids recovered from soils have been widely studied and the assignments of the most important bands are summarized in Table 4, together with the references. The moisture adsorbed by the sample can give bands at 3400 and 1630 c m - ~: this is the reason why the assignment of these bands is difficult. Other absorption bands are present at wavenumbers in the range 1500-1000cm -I and here again their assignment is rather difficult, owing to the complexity of the spectra in this region. The bands near 1450cm-~ could be assigned to aliphatic structures (Bellamy, 1975) and to phenolic OH groups (Stevenson & Goh, 1971; Russell et al., 1983). The classification of IR spectra proposed by Stevenson & Goh (1971) can be of value in discussing the IR analysis of our HA and FA samples. IR spectra of soil HA and FA have been classified into three general types. Type I: these spectra show strong absorption bands near 3400, 2930, 1720, 1600 and 1200cm -1, the intensities of the bands at 1720 and 1600 c m - 1 being comparable.

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Ezio Roletto, Maria Paola Luda

TABLE 4 Infrared Spectra: Assignment of Absorption Bands of HA and FA Extracted from Soils Wavenumber (cm - 1 )

Assignment

3400 Moisture;H bonded OH groups of carboxyls, phenols and broad band alcohols 2930 Aliphatic C--H stretching 1720 C : O groups in carboxyls, ketones and aldehydes strong band 1630-1600 Moisture;aromatic C~-C vibrations; C~-~-Ogroups in quinones; C : O in conjugated ketones 1540 Peptidelinkage in proteinaceous materials 1510 Skeletalin-plane vibrations of aromatic rings 1200 C--O stretching; OH deformation of carboxyls broad band 1050 C--O stretching of polysaccharides under 1000 C--H out-of-plane vibrations of aromatic rings

Reference

1-2-3 1-2-3 1-2-3 1-2-3 1-3 2 1 1 2

1: Stevenson & Goh (1971). 2: Theng et aL (1967). 3: Bellamy (1975). Type II: spectra of this type are characterized by strong absorption at 1720cm-1; absorption in the 1600 region is less strong and centered near 1630 c m - I Type III: these spectra show not only the major bands shown by types I and II, but also relatively strong bands at 1540 and 1050cm -~. Soil H A generally show type I or II spectra, whilst soil F A exhibit type II or III spectra. Hydrolysis of soil humic fractions, characterized by type III spectra, destroyed proteins and carbohydrates of the sample and produced residues whose spectra were very similar to those of type I or type II (Stevenson & Gob, 1971). HA12 and HA30 spectra show some similarities with those of humic acids recovered from soil (Stevenson & Goh, 1971, Fig. 1, spectra A and B). More specifically, they show all the absorption bands o f spectra classified as type I; in addition, our spectra are more structured in the 'fingerprint' region ( < 1300 cm-1); the band at 1510 c m - 1 is not present in the spectra of Stevenson and G o h and the absorption band at 1720cm-1 is less strong. FA12 and FA30 spectra show the more important features of type II spectra, but the band at 1640cm-1 is more important in our samples than in fulvic acids recovered from soil (Stevenson & Goh, 1971, Fig. 2, spectra B and C). Our spectra are quite structured in the low frequencies region, and the bands at 1510 and 740cm -I, which are not found in the spectra of Stevenson and Goh, could probably be attributed to aromatic structures.

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Infrared spectra of HA extracted from poplar bark at different humification stages show no important differences. On the other hand, IR spectra of FA12 and FA30 fractions show some differences. More specifically, FA30 spectra exhibit stronger absorption bands than FA12 at 1720 and near 2600cm -~. The latter is a shallow band assigned to intermolecular H-bonding of carboxylic groups (Bellamy, 1975). Both saturated ketones and carboxylic acids absorb at 1720 cm-~, but since the functional groups analysis shows a decreasing C~---O and an increasing C O O H content as the humification period increases, the major contribution to the absorption in this region should be attributed to C ~ O groups in carboxyls. Absorption at 1720cm-1 could be almost completely assigned to carboxylic acid groups, but absorption at 1630cm-1 could be attributed to hygroscopic moisture and to different functional groups, so that we cannot quantitatively correlate the spectral data with those obtained by chemical analysis of functional groups. Nevertheless, the intensity of COOH bands is greater in FA than in HA, confirming the data obtained by the titration techniques.

ACKNOWLEDGEMENT The work reported in this paper was carried out with the financial support of the Ministero della Pubblica Istruzione.

REFERENCES Bellamy, L. J. (1975). The infra-red spectra of complex molecules. John Wiley, New York. Hatcher, P. G., Maciel, G. E. & Dennis, L. W. (1981). Aliphatic structures of humic acids; A clue to their origin. Org. Geochem., 3, 43-8. Roletto, E. & Ottino, P. (1984). Analytical characterization of humic substances from composted lignocellulosic residues. Agric. Wastes, 11, 181-95. Roletto, E., Barberis, R. & Zelano, V. (1982). Gel filtration and absorption spectroscopic investigation on humic substances from organic fertilizers. Plant and Soil, 66, 383-90. Roletto, E., Chiono, R. & Barberis, E. (1985). Investigation on humic matter from decomposing poplar bark. Agric. Wastes, 12, 261-72. Russell, J. D., Vaughan, D., Jones, D. & Fraser, A. R. (1983). An IR spectroscopic study of soil humin and its relationship to other soil humic substances and fungal pigments. Geoderma, 29, 1-12. Schnitzer, M. (1978). Humic substances: chemistry and reactions. In: Soil organic matter. (Schnitzer, M. & Khan, S. U. (Eds)). Elsevier Sci. Pub., Amsterdam, 1-64.

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Schnitzer, M. & Khan, S. U. (1972). Humic substances in the environment. Marcel Dekker, Inc., New York, 37-44. Stevenson, F. J. & Gob, K. M. (1971). Infrared spectra of humic acids and related substances. Geochhn. Cosmochim. Acta, 35, 471-83. Stevenson, F. J. & Gob, K. M. (1972). Infrared spectra of humic and fulvic acids and their methylated derivatives: Evidence for non-specificity of analytical methods for oxygen-containing functional groups. Soil Sci., 113, 334-45. Theng, B. K. G., Wake, J. R. H. & Posner, A. M. (1967). The humic acids extracted by various reagents from a soil. II. Infrared. visible and ultra-violet absorption spectra. J. Soil Sci., 18, 349-363. Thurman, E. M., Malcom, R. L. & Aiken, G. R. (1978). Prediction of capacity factors for aqueous organic solutes adsorbed on a porous acrylic resin. Anal Chem., 50, 775-9.