- Email: [email protected]
Biochemical degradation of soil humic acids and fungal melanins
Organic Geochemistry 33 (2002) 347–355 www.elsevier.com/locate/orggeochem
Biochemical degradation of soil humic acids and fungal melanins Yu.A. Zavgorodnyayaa,*, V.V. Deminb, A.V. Kurakova a
Moscow State University, Soil Science Department, 119899, Vorob’evy Gory, Moscow, Russia b Institute of Soil Science MSU-RAS, 119899, Vorob’evy Gory, MSU, Moscow, Russia
Abstract Studies were conducted to compare properties and biodegradation of fungal melanins from Aspergillus niger and Cladosporium cladosporiodes with those of humic acids from soils and brown coal. Compared to the humic acids the fungal melanins contained more functional groups, were less hydrophilic and had relatively high molecular weights. Under the conditions of incubation the melanins were found to be more readily degradable than the humic acids studied. The changes in elemental composition, optical parameters and the decrease of molecular weight, observed for both fungal melanins during degradation, made them more similar to soil humic acids. # 2002 Published by Elsevier Science Ltd.
1. Introduction Soil humic acids constitute a very important fraction of soil humus due to their physical, chemical and biological effects on soil and whole ecosystem. Different organic compounds of microbial and plant origin, being the source of aromatic and aliphatic units, find their way into the soil and participate in the formation of humic substances (Stevenson, 1982; Orlov, 1990). Melanins—dark-colored high molecular weight pigments, produced by many soil fungi—stand out from the other substances, which are considered to be precursors of humic acids. Being the products of intracellular synthesis, melanins at the same time are found to be remarkably similar in some chemical and physico-chemical properties to soil humic acids, which are believed to be formed from the constituent units (such as lignoid and phenolic compounds combined with carbohydrates and peptides) extracellulary and therefore in different conditions. Though fungal melanins widely vary in their properties,
* Corresponding author. Tel.: +7-095-9395164; fax: +7095-9393774. E-mail address: [email protected] (Yu.A. Zavgorodnyaya).
they have certain similarities to humic acids in term of their behavior in solvents, elemental composition, exchange acidity, UV and visible spectra, infra-red and 13 C nuclear magnetic resonance spectra, pyrolysis-mass spectra, phenolic compounds recovered upon Na-amalgam reductive degradation, amino acids released upon hydrolysis with 6 N HCl and low polysaccharide content (Kang and Felbeck, 1965; Martin and Haider, 1969; Zaprometova et al., 1971; Volnova and Mirchink, 1972; Ellis and Griffiths, 1974; Filip et al., 1976; Valmaseda et al., 1989; Knicker et al., 1995). Most of the studies, concerning the fungal contribution to the process of accumulation of humic acid-like polymers in soil, are only based on the above mentioned similarities between humic acids and fungal melanins, extracted from the mycelium. Some investigators have theorized that the bulk of unaltered melanins, produced by different soil fungi, form the most stable fractions of soil humus known as humic acids (Kang and Felbeck, 1965; Zviagintsev and Mirchink, 1986). However, the role of melanins in humus accumulation depends predominantly on their resistance to degradation by soil microorganisms. Resistance to biodegradation is one of the most important properties of soil humic substances. If fungal pigments do actually make a significant contribution to the humic fractions of the soil, their
0146-6380/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0146-6380(01)00165-6
348
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
decomposition rates have to be comparable with those of humic acids. When comparing resistance of humic acids and melanins to biodegradation, it is important to take into account not only the rates of their decomposition to simple inorganic substances such as CO2, but also the possible alteration of the chemical structure of their molecules due to microbial activity. Independently of the extent of mineralization, the changes in chemical properties indicate biological degradation of the polymer. The purpose of the present work was to compare the properties and biochemical degradation of soil humic acids from sod-podzol soil and chernozem, brown coal humic acid and melanins of soil fungi Aspergillus niger and Cladosporium cladosporiodes under conditions of a laboratory incubation experiment. The brown coal humic acid was selected as an inert sample, which had undergone both transformation during diagenesis and drastic chemical treatment during the industrial extraction from the coal, and which was expected therefore to be highly resistant to microbial degradation. The specific objectives of the work were: (1) to determine mineralization rate of humic acids and fungal melanins and (2) to study physico-chemical properties of humic acids and melanins and changes of these properties during 3 months of biodegradation.
2. Material and methods 2.1. Extraction of the humic acids and fungal melanins Humic acids were obtained by extraction with 0.1 M NaOH from air-dried soils ground to 1 mm. Humic acids were precipitated by acidification to pH 2 with HCl and purified by desalting with 0.4 M NaCl. For brown coal humic acid a commercial sodium humate, purified and transferred to H+-form, was used. The method used for culturing fungal mycelium was similar to that described by Linhares and Martin (1978). Briefly, the fungal cultures were grown in a mineral medium, containing (g l 1) 2.0 K2HPO4; 0.2 MgSO47H2O; 0.1 KCl; 2.0 NH4NO3; 30 glucose. Medium (200–250 ml) were placed in 900-ml Roux bottles and sterilized under pressure for 30min. The Roux bottles were incubated at 25 C on their flat sides until the pads became black from the melanin. After incubation the pads were removed from the culture medium, washed with distilled water and freeze-dried. Melanins were extracted from mycelium with 0.1 M NaOH, precipitated with HCl, purified and dried at 40 C. Ash content of all samples was <5% after purification. 2.2. Incubation experiments Samples of 100 mg of melanin or humic acid were thoroughly mixed with 20 g of quartz sand, 200 mg of
14
C-glucose was added, and the mixture was inoculated with 1 ml of aqueous extract containing an indigeneous microbial population of chernozemic soil. When mixed microbial population are used, the effect of individual soil microorganisms on organic matter decomposition is of minor importance (Dormaar, 1975). Glucose, considered as a carbon source easily decomposed by microorganisms, was added to stimulate microbial activity in the mixture and to prime decomposition of melanins and humic acids (Bingeman et al., 1953). Incubations were performed at 25 C in triplicate in 100 ml screw-capped jars, at 60% of field capacity. After 3 months of incubation melanins and humic acids were extracted from the mixture with 0.1 M NaOH and analyzed. Melanin and humic acid C lost by mineralization was the total amount of CO2-C evolved from the incubated mixture minus the amount of 14C-CO2 respired from decomposed 14C-glucose and 14C-labeled microbial tissues. The CO2 evolved was trapped in 8 ml 0.5 M KOH, placed in a plastic vial at the surface of the incubated mixture. To determine the amount of total CO2, at selected intervals, the vial was replaced from the jar and excess base was titrated with 1.0 M H2SO4 (Ivannikova, 1992). To determine the 14C-CO2 amount, 1 ml of KOH before titration was placed into a scintillator vial, 1 ml of scintillating mixture GS-13 was added and the radioactivity (count min 1) was measured on a LKB RackBeta 1217 liquid scintillation counter. Values of counts per minute were then converted into decays per minute using an internal standard method (L’Annunziata, 1979). 2.3. Analytical methods The C, H and N contents of initial and degraded humic acids and fungal melanins were determined using a Carlo-Erba 1106 CHNS elemental analyzer (T=1000 C). Functional groups in humic acids and melanins were determined by potentiometric titration. Preparations, diluted in 0.05 M NaOH (pH 11.7, concentration=0.5 mg/ml) were titrated with 0.1 M HCl at 25 C, ionic strength 0.1 (KCl) under permanent N2 flow. pH were recorded using a Horiba pH-meter after the value had remained stable for 1 min. To find the beginning and the end points of humic acids and melanins titration Gran’s analysis was used. Dissociation constants (pKa) were obtained as described by Takamatsu and Yoshida (1978). Infrared spectra were recorded on a Vector 22 spectrophotometer (Bruker, Germany) using KBr pellets, obtained by pressing, under vacuum, uniformly prepared mixtures of 1 mg dried preparation and 400 mg KBr. The UV and visible spectra of humic acids and melanins solutions were recorded at pH 11–12 on a Shimadzu
349
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
UV-260 spectrophotometer. The optical density (E4) was measured at wavelength 465nm, the extinctions, E 465 nm and E 665 nm, were used to calculate E4/E6 ratio. Molecular weight distributions of the melanins were obtained by gel filtration, using a Pharmacia column K 970 with Sephadex G-100 gel. Solution of 0.025 M Tris–HCl buffer (pH 8.1), 0.1% sodium dodecyl sulfate, 0.5 M NaCl and 0.02% NaN3 was used as an effluent. The elution rate was 3 ml cm 2 h 1. The optical density of the eluted solution was measured at wavelength 280 nm using a 2238 Uvicord S II detector (LKB Sweden). All samples were purified from low molecular weight contaminants by elution through a Sephadex G-10 column. Molecular weights of the fractions were calculated, using the formula developed by Determan (1971) for globular proteins, which was the following for Sephadex G-100 gel: log10M=5.941–0.847(Ve/V0), where M— molecular weight in kDa; Ve—elution volume in ml and V0—void volume in ml. Percentage content of each fraction was determined by calculating the area of the corresponding peak on the curve. The peak areas were calculated using ‘‘triangle method’’, developed for unresolved chromatographic peaks (Petzev and Kotzev, 1987). The distribution of hydrophobic and hydrophilic sites in humic acids and melanins was determined on a 1010 Amicon column, filled with octyl-sepharose CL4B gel. Elution was made at a rate of 10 ml cm 2 h 1 firstly with 0.05 M Tris–HCl buffer (pH 8.2) and then with 0.05 M Tris–HCl (pH 8.2)+0.3% Na-dodecylsulfate buffer using a step buffer change gradient. The step gradient was selected to divide the samples into two well-resolved fractions: fraction with predominance of hydrophilic properties (hydrophilic fraction) and fraction with predominance of hydrophobic properties (hydrophobic fraction). The content of the hydrophilic fraction was determined taking into consideration the corresponding peak area on the chromatogram.
(1982) for different soil systems. Subsequently mineralization of the unlabelled C from melanins and humic acids started, and for the next 10 weeks only 10% of the applied 14C evolved via decomposition and reutilization of 14C-labelled microbial tissues. After 91 days between 82 and 89% of the added 14C had been respired as CO2. All decomposition curves of the humic acids and fungal melanins follow an exponential pattern (Fig. 1). A relatively high mineralization rate for all samples was observed during the 1st month of incubation, after which the process decreased considerably. The decline of the mineralization process is known to be connected to the utilization of the labile part of melanins and humic acids and the formation of toxic products, that suppress microbial activity in the incubated mixture (Alexandrova, 1980). The decomposition rate of the humic acid from chernozem was 8% and for humic acid from sod-podzol soil—12% after 3 months of incubation. Coal humic acid was more resistant to biodegradation and showed only 3% loss of applied C for 3 months. Fungal melanins appeared to be more readily biodegradable: 22% C for A. niger and 25% C for C. cladosporiodes were released as CO2 during the experiment (Fig. 1). These values were comparable with those reported for some humic acids (Linhares and Martin, 1978; Alexandrova, 1980) and some melanins (Wolf and Martin, 1976; Boudot et al., 1989). The assessment of the incorporation of 14C from microbial biomass into humic acids and melanins (Table 1) showed that the highest level of incorporation was observed after 2 weeks of incubation (5–7% of applied 14C). After 3 months 14C was found only in sodpodzol humic acid and A. niger melanin. 3.2. Changes of physico-chemical properties of humic acids and fungal melanins during incubation Humic acid and melanin elemental composition, atomic ratios, functional group content and hydrophilic properties are shown in Table 2.
3. Results and discussion 3.1. Mineralization of humic acids and fungal melanins The CO2 emission from the incubated mixtures started on the 3rd day after inoculation. The lag-period observed was related to the slowing of microbial growth, caused by the high initial content of glucose untypical for usual soil conditions (Panikov et al., 1982). During the first two weeks of incubation mineralization of 14C-glucose and growth of 14C-labelled microbial biomass occurred. During this period about 70% of the applied 14C evolved as CO2. Similar mineralization rates for 14C-glucose (60–80% over 14 days) were reported by Cheshire et al. (1969), Dormaar (1975) and Zunino et al.
Table 1 Percentage of applied during incubation
14
C found in humic acids and melanins Days of uncubation
Sample
0
16
47
91
Sod-podzol humic acid Chernozem humic acid Brown coal humic acid Aspergillus niger melanin Cladosporium cladosporiodes melanin
– – – – –
6.1 5.0 4.5 7.0 5.0
1.9 – 1.2 3.1 –
2.2 – – 3.8 –
350
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Fig. 1. Mineralization rate of humic acids and fungal melanins (cumulative values). =confidence limits at 95% level.
Table 2 Analytical characteristics of humic acids and fungal melanins Sample
Content (at.%)
Atomic ratios
Functional groups
Content of hydrophilic
C
H
N
Oa
H/C
O/C
C/N
(mM( )/g)
fraction (%)
Sod-podzol humic acid
37.3b 36.5c
41.3 41.7
2.7 2.5
18.7 19.3
1.11 1.14
0.50 0.53
13.8 14.4
7.48 –
53 –
Chernozem humic acid
41.8b 39.0c
35.3 36.9
3.2 3.2
19.7 20.9
0.84 0.95
0.47 0.54
13.1 12.1
7.00 –
56 –
Brown coal humic acid
37.2b 39.2c
31.4 35.2
0.6 0.7
30.9 24.9
0.84 0.90
0.83 0.64
62.0 60.1
5.60 –
49 –
A. niger melanin
37.3b 38.2c
41.6 39.5
3.1 2.9
18.0 19.4
1.12 1.03
0.48 0.51
12.0 13.1
9.00 –
–
C.cladosporiodes melanin
33.9b 35.9c
45.2 39.7
4.0 4.5
16.9 20.0
1.33 1.11
0.50 0.58
8.5 8.1
10.56 –
14 –
a b c
By difference. Before incubation. After incubation.
Results of potentiometric titration of humic acids and fungal melanins in the pH region 4.0–10.5 show the presence of three types of functional groups: (1) with pKa value 4.4–4.7 (strong carboxylic groups, being titrated in pH range 3.5–7.0); (2) with pKa value 7.5–8.1 (weak carboxylic and phenolic, amine groups, being titrated in pH region 7.0–8.5); (3) with pKa value 9.6–9.9
(phenolic groups, being titrated in pH region over 8.5). Compared with the coal and soil humic acids, total content of functional groups of both fungal melanins was much higher, probably being evidence of higher reactivity of fungal melanins (Table 2). The infrared spectra of the melanins were found to be similar to those of the humic acids. Melanin from A.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
niger resembled IR-spectra of chernozem humic acid. IR-spectra of C. cladosporiodes melanin had some similarities with those of sod-podzol humic acid (Fig. 2). The elemental composition of the A. niger melanin was similar to those of the humic acids (Table 2). Melanin from C. cladosporiodes had a relatively low O content and high N content (4 at.%), compared with the soil and coal humic acids. The high N content was also reflected in the IR-spectrum of C. cladosporiodes melanin by an intense band at 1530 cm 1 (attributed to amide I protein linkages), indicating significant amounts of peptides. The band at 1530 cm 1 occurred with moderate intensity also in the spectrum of sod-podzol humic acid (Fig. 2). The H/C ratio, which is usually used to assess the contribution of aliphatic chains to the molecular structure, was the highest for the melanin from C. cladosporiodes and the bands at 2920–2860 cm 1 (aliphatic C– H stretching) were the most intense on the IR-spectrum of C. cladosporiodes melanin. According to the H/C ratio a high degree of aromaticity was obtained for the chernozem and brown coal humic acids (Table 2). This was confirmed by low absorption at 2920–2860 cm 1 and high absorption at 1610 cm 1, reflecting the abundant presence of C=C bonds, on the IR-spectra of both humic acids (Fig. 2). There were no substantial changes in the elemental composition of soil humic acids after 3 months of incubation, but a minor trend towards a decrease of C content and an increase of H content was observed
Fig. 2. Infrared spectra of humic acids and fungal melanins: (1) brown coal humic acid; (2) chernozem humic acid; (3) sodpodzol humic acid; (4) Cladosporium cladosporiodes melanin; (5) Aspergillus niger melanin.
351
(Table 2). This indicates, that slight loosening of the humic acid structure occurs due to microbial activity, and humic acid molecules become less condensed (Chernikov, 1992). The O content of the humic acid from the brown coal decreased after the experiment and the optical density of the humic acid decreased simultaneously (Table 3). This suggests a possible relationship between these two characteristics. For fungal melanins a decrease of H content and an increase of C content was observed after 3 months, that may be caused by an increase of the aromaticity of the pigments (Table 2). The results of hydrophobic chromatography are shown in Table 2. All humic acids consisted of two fractions—hydrophobic and hydrophilic—of almost equal abundances. Chernozem humic acid was the most hydrophilic. C. cladosporiodes melanin probably contained more hydrophobic sites than humic acids, based on the low content of hydrophilic fraction (Table 2). A. niger melanin sorbed on the octyl-sepharose gel, due to unspecific interactions. Raw precipitates of A. niger melanin are known to be highly hydrophilic, but lose hydrophilic properties after drying (Nicolaus, 1968). UV and visible absorption curves of humic acids were featureless (Fig. 3), with the exception of sod-podzol humic acid, which had a shoulder-like maximum in the 450 nm region and small maximum at 620 nm, reflecting the presence of Pg-pigment. Formation of such greencoloured pigments (so-called ‘‘Pg-fraction’’ of humic acids) in soil is due to the presence of certain fungi, such as Cenococcum graniforme, which synthesize a polynuclear quinoid pigment considered to be a perylene derivative (Sato and Kumada, 1967; Sato, 1976). Melanin from A. niger showed two broad, distinct maxima at 300 and 420 nm of unknown origin. The presence of such maxima may be connected to the contamination of the melanin with some non-melanin pigments, which contribute, according to Nicolaus (1968), up to 10% of the melanin’s weight. Spectral parameters (E4 extinction and E4/E6 ratio) of the fungal melanins (Table 3) were in the range reported for humic acids of different origin (Stevenson, 1982). However, the extinction coefficient of the initial melanin from C. cladosporiodes was much lower than for all humic acids studied and it was more similar to that of humic acids with a small degree of maturity and diagenesis (Chen et al., 1977). The E4-value of the A. niger melanin was the highest, but probably this value was overestimated due to the broad maximum at 420 nm. There were almost no changes in the optical properties of soil humic acids during incubation. At the same time changes of such properties for melanins were noteworthy (Table 3). After degradation the extinction of the A. niger melanin decreased, while the extinction of the degraded C. cladosporiodes melanin was three times greater than that of the initial pigment. At the same time the form of the UV-visible absorption curves of both
352
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Table 3 Changes of molecular weights and spectroscopic parameters of humic acids and fungal melanins during incubation Sample
Days of incubation
Spectroscopic parameters
Percentage of molecular weight fractions (kDa)
E4
E4/E6
>150
70
10
Sod-podzol humic acid
0 16 47 91
0.075 0.070 0.074 0.077
4.6 4.6 4.7 4.6
7 10 7 4
21 18 16 13
72 72 77 81
Chernozem humic acid
0 16 47 91
0.115 0.101 0.115 0.117
4.0 4.0 4.1 4.0
3 5 2 1
13 1 2 95
84 83 89 94
Brown coal humic acid
0 16 47 91
0.144 0.125 0.128 0.134
5.8 4.2 4.8 5.2
2 5 4 2
4 4 4 3
94 91 92 95
A. niger melanin
0 16 47 91
0.176 0.141 0.153 0.157
2.9 2.9 2.9 3.1
8 10 8 6
30 32 27 26
62 58 65 68
C. cladosporiodes melanin
0 16 47 91
0.030 0.041 0.080 0.096
3.6 3.6 3.7 3.7
40 42 29 22
42 44 24 28
18 14 47 50
Fig. 3. UV and visible spectra of humic acids and fungal melanins: (1) sod-podzol humic acid; (2) chernozem humic acid; (3) brown coal humic acid; (4) Aspergillus niger melanin; (5) Cladosporium cladosporiodes melanin.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
353
Fig. 4. Changes of molecular weight distributions of humic acids and fungal melanins during biodegradation.
melanins, and those of the humic acids, remained unchanged after 3 months of incubation. Also the E4/E6 ratios stayed almost constant for the duration of the experiment.
An increase of optical density together with an increase of O content of C. cladosporiodes melanin suggests that during degradation oxidizing reactions occur and that an additional amount of chromophoric groups
354
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
had formed in the melanin molecules. These O-containing chromophoric groups are probably formed by oxidation of the polypeptide or polysaccharide chains without cleavage of the aromatic rings. Such oxidative degradation reactions of the side chains in the melanin molecules result in a significant decrease in the molecular weight and in the simultaneous increase of the E4 value of C. cladosporiodes melanin. A. niger melanin is probably degraded by the destruction of the aliphatic chains with alternate double and single bonds, linked to the aromatic rings. The presence of these chains, linking aromatic rings, is considered to be one of the factors, which causes high optical density of humic acids (Orlov, 1990). The cleavage of the aliphatic chains with alternate double and single bonds reduces the extinction coefficient of A. niger melanin. The increase of the aromaticity of both pigments after incubation (Table 2) is also connected with the oxidative degradation of aliphatic components in the melanins. The molecular weight distributions of the initial melanins and humic acids were dissimilar (Fig. 4). Distribution curves of melanins had narrow, well-resolved peaks, that are typical for mixtures of individual components with high molecular weights. Similar distributions are obtained, for example, for mixtures of different proteins. In contrast humic acids, being polymolecular compounds, revealed a very different distribution of molecular sizes with relatively wide peaks. The molecular weight distributions of soil humic acids changed after incubation; the peaks became more symmetrical, indicating, that the fractions of these humic acids became less polydispersed. At the same time the molecular weight distributions of the melanins showed peaks widening (Fig. 4). On the gel-chromatographic curves of all tested samples three fractions were observed: (1) a maximum molecular weight above 150 kDa, (2) around 70 kDa and (3) around 10 kDa (Fig. 4). In Table 3 the percentage of each fraction is shown. The relative contents of the low molecular weight fraction (Mw 10 kDa) of the initial fungal pigments were lower than those found for the humic acid studied, especially of C. cladosporiodes. This finding is in accordance with the results obtained by Valmaseda et al. (1989) for melanins of fungal species, isolated from two Umbric Cambisols; molecular weights of those melanins were higher than for the humic acids of the corresponding soils. In our previous study melanins, obtained from 15 strains of soil fungi, were also found to have a relatively low content (10– 40%) of the fraction with Mw 10 kDa when compared to soil humic acids (Zavgorodnyaya et al., 2001). After 2 weeks of incubation, the high molecular weight fraction (Mw > 150 kDa) had increased for all polymers studied. These results, together with the high content of 14C in all polymers (Table 1), indicated that some organic compounds of microbial origin (large
fragments of peptides) had been incorporated into the humic acids and melanins after 2 weeks. After 6 weeks decrease of the high molecular weight fraction and of the 14C content suggests that these incorporated compounds had been utilized by microorganisms. After 3 months incubation the content of the low molecular weight fraction (Mw 10 kDa) increased and the content of the high molecular weight fractions decreased for all polymers with the exception of brown coal humic acid, which displayed insignificant changes (Table 3). For the incubated samples the changes of the initial distribution of the molecular weight fractions suggest different rates of fraction degradation. The high molecular weight fractions appeared to be less resistant to biodegradation and to form fragments with low molecular weight, which enlarged the fraction with Mw 10 kDa. The relative rate of degradation of the Mw 10 kDa fraction was lower, in comparison with the high molecular weight fractions, as a result accumulation of the low molecular weight fraction was observed for soil humic acids and fungal melanins. The most intense degradation was observed for the high molecular weight fractions of C. cladosporiodes melanin (Table 3). These fractions were probably enriched in the compounds (such as long peptide or carbohydrate chains), which were removed from the melanin molecule by microbial oxidative destruction.
4. Conclusion Some differences in functional groups content, spectral parameters and molecular weights between initial fungal melanins and humic acids were observed. However, for C. cladosporiodes melanin these differences were prominent, whereas A. niger melanin appeared to be more similar to the soil humic acids. During incubation fungal melanins were both intensively degraded by soil microorganisms, and after 3 months of degradation, became more like humic acids due to removal of aliphatic components, changes in elemental composition and in optical parameters and decrease of molecular weight. Humic acids under the same incubation conditions, even being partially mineralized, did not change their properties significantly. These results suggest that if melanin pigments of soil fungi, at least for these two species, are to become significant contributors to the stable soil organic fraction, they have to undergo a number of chemical transformations, drastically affecting the melanin properties. For further investigations of pigment contribution to humic acid accumulation, melanin studies of a large number of fungal species, along with quantitative assessment of annual melanin input to the soil, will be essential.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Acknowledgements The authors are grateful to the Russian Foundation for Basic Research (project Nos. 99-04-48007 and 00-1597856) and the Russian Federal Scientific Program (project 4.1.3) for support of this work. References Alexandrova, L.N., 1980. Soil Organic Matter and Processes of Its Transformation. Nauka, Leningrad. Bingeman, C.W., Varner, J.E., Martin, W.P., 1953. The effect of addition of organic materials on the decomposition of an organic soil. Soil Science Society of America Proceedings 17, 34–37. Boudot, J.-P., Bel Hadi Brahim, A., Steiman, R., Seigle-Murandi, F., 1989. Biodegradation of synthetic organo-metallic complexes of iron and aluminium with selected metal to carbon ratios. Soil Biology and Biochemistry 21, 961–966. Chen, Y., Senesi, N., Schnitzer, M., 1977. Information provided on humic substances by E4-to-E6 ratios. Soil Science Society of America Journal 41, 352–358. Chernikov, V.A., 1992. Transformation of humic acids as affected by autochtonous microflora. Russian Soil Science 3, 69–77. Cheshire, M.V., Mundie, C.M., Shepherd, H., 1969. Transformation of 14C glucose and starch in soil. Soil Biology and Biochemistry 1, 117–130. Determan, G., 1971. Gel-chromatography. Mir, Moscow. Dormaar, J.F., 1975. Susceptibility of organic matter of chernozemic Ah horizons to biological decomposition. Canadian Journal of Soil Science 55, 473–480. Ellis, D.H., Griffiths, D.A., 1974. The location and analysis of melanins in the cell walls of some soil fungi. Canadian Journal of Microbiology 20, 1379–1386. Filip, Z., Semotan, J., Kutilek, M., 1976. Thermal and spectrophotometric analysis of some fungal melanins and soil humic compounds. Geoderma 15, 131–142. Ivannikova, L.A., 1992. Using of absorption method for detecting of the natural CO2 efflux from a soil. Russian Soil Science 6, 133–139. Kang, K.S., Felbeck, G.T., 1965. A comparison of the alkaline extract of tissues of Aspergillus niger with humic acids from three soils. Soil Science 99, 175–181. Knicker, H., Almendros, G., Gonzalezvila, F.J., Ludemann, H.D., Martin, F., 1995. 13C and 15N NMR analysis of some fungal melanins in comparison with soil organic matter. Organic Geochemistry 23, 1023–1028. L’Annunziata, M.F., 1979. Radiotracers in Agricultural Chemistry. Academic Press, London.
355
Linhares, I.F., Martin, J.P., 1978. Decomposition in soil of the humic acid-type polymers (melanins) of Eurotium echinulatum, Aspergillus glaucus sp. and other fungi. Soil Science Society of America Journal 42, 738–743. Martin, J.P., Haider, K., 1969. Phenolic polymers of Stachybotrys atra, Stachybotrys chartarum and Epicoccum nigrum in relation to humic acid formation. Soil Science 107, 260–270. Nicolaus, R.A., 1968. Melanins. Hermann, Paris. Orlov, D.S., 1990. Humus Acids and the General Theory of Humification. MSU Press, Moscow. Panikov, N.S., Abu El-Naga, S.A., Zvyagintsev, D.G., 1982. Kinetics of glucose decomposition in soils. Soviet Soil Science 8, 70–77. Petzev, N., Kotzev, N., 1987. Manual on Gas Chromatography. Mir, Moscow. Sato, O, 1976. The chemical nature of components of Pg (green fraction of P-type humic acid). Soil Science and Plant Nutrition 22, 485–488. Sato, O., Kumada, K., 1967. The chemical nature of P-type humic acid. Soil Science and Plant Nutrition 13, 121–122. Stevenson, F.J., 1982. Humus Chemistry. Genesis, Composition, Reactions. Wiley and Sons, New York. Takamatsu, T., Yoshida, T., 1978. Determination of stability constants of metal-humic acid complexes by potentiometric titration and ion-selective electrodes. Soil Science 125, 377– 386. Valmaseda, M., Martinez, A.T., Almendros, G., 1989. Contribution by pigmented fungi to P-type humic acid formation in two forest soils. Soil Biology and Biochemistry 21, 23–28. Volnova, A.I., Mirchink, T.G., 1972. Fractionation of darkcolored fungi containing a green fraction. Soviet Soil Science 11, 140–145. Wolf, D.C., Martin, J.P., 1976. Decomposition of fungal micelia and humic-type polymers containing 14carbon from ring and side-chain labeled 2,4-D and chlorpropham. Soil Science Society of America Journal 40, 700–704. Zaprometova, K.M., Mirchink, T.G., Orlov, D.S., Yukhnin, A.A., 1971. Characteristics of black pigments of the darkcolored soil fungi. Soviet Soil Science 7, 22–30. Zavgorodnyaya, Yu.A., Demin, V.V., Kurakov, A.V., Biryukova, O.N., 2001. Characteristic of physico-chemical properties of fungal melanins. Moscow State University Herald, Series 17: Soil Science 3, 39–43. Zunino, H, Borie, F., Aguilera, S., Martin, J.P., Haider, K., 1982. Decomposition of 14C-labelled glucose, plant and microbial products and phenols in volcanic ash-derived soils of Chile. Soil Biology and Biochemistry 14, 37–43. Zviagintsev, D.G., Mirchink, 1986. On the nature of soil humic acids. Soviet Soil Science 5, 68–75.
Biochemical degradation of soil humic acids and fungal melanins Yu.A. Zavgorodnyayaa,*, V.V. Deminb, A.V. Kurakova a
Moscow State University, Soil Science Department, 119899, Vorob’evy Gory, Moscow, Russia b Institute of Soil Science MSU-RAS, 119899, Vorob’evy Gory, MSU, Moscow, Russia
Abstract Studies were conducted to compare properties and biodegradation of fungal melanins from Aspergillus niger and Cladosporium cladosporiodes with those of humic acids from soils and brown coal. Compared to the humic acids the fungal melanins contained more functional groups, were less hydrophilic and had relatively high molecular weights. Under the conditions of incubation the melanins were found to be more readily degradable than the humic acids studied. The changes in elemental composition, optical parameters and the decrease of molecular weight, observed for both fungal melanins during degradation, made them more similar to soil humic acids. # 2002 Published by Elsevier Science Ltd.
1. Introduction Soil humic acids constitute a very important fraction of soil humus due to their physical, chemical and biological effects on soil and whole ecosystem. Different organic compounds of microbial and plant origin, being the source of aromatic and aliphatic units, find their way into the soil and participate in the formation of humic substances (Stevenson, 1982; Orlov, 1990). Melanins—dark-colored high molecular weight pigments, produced by many soil fungi—stand out from the other substances, which are considered to be precursors of humic acids. Being the products of intracellular synthesis, melanins at the same time are found to be remarkably similar in some chemical and physico-chemical properties to soil humic acids, which are believed to be formed from the constituent units (such as lignoid and phenolic compounds combined with carbohydrates and peptides) extracellulary and therefore in different conditions. Though fungal melanins widely vary in their properties,
* Corresponding author. Tel.: +7-095-9395164; fax: +7095-9393774. E-mail address: [email protected] (Yu.A. Zavgorodnyaya).
they have certain similarities to humic acids in term of their behavior in solvents, elemental composition, exchange acidity, UV and visible spectra, infra-red and 13 C nuclear magnetic resonance spectra, pyrolysis-mass spectra, phenolic compounds recovered upon Na-amalgam reductive degradation, amino acids released upon hydrolysis with 6 N HCl and low polysaccharide content (Kang and Felbeck, 1965; Martin and Haider, 1969; Zaprometova et al., 1971; Volnova and Mirchink, 1972; Ellis and Griffiths, 1974; Filip et al., 1976; Valmaseda et al., 1989; Knicker et al., 1995). Most of the studies, concerning the fungal contribution to the process of accumulation of humic acid-like polymers in soil, are only based on the above mentioned similarities between humic acids and fungal melanins, extracted from the mycelium. Some investigators have theorized that the bulk of unaltered melanins, produced by different soil fungi, form the most stable fractions of soil humus known as humic acids (Kang and Felbeck, 1965; Zviagintsev and Mirchink, 1986). However, the role of melanins in humus accumulation depends predominantly on their resistance to degradation by soil microorganisms. Resistance to biodegradation is one of the most important properties of soil humic substances. If fungal pigments do actually make a significant contribution to the humic fractions of the soil, their
0146-6380/02/$ - see front matter # 2002 Published by Elsevier Science Ltd. PII: S0146-6380(01)00165-6
348
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
decomposition rates have to be comparable with those of humic acids. When comparing resistance of humic acids and melanins to biodegradation, it is important to take into account not only the rates of their decomposition to simple inorganic substances such as CO2, but also the possible alteration of the chemical structure of their molecules due to microbial activity. Independently of the extent of mineralization, the changes in chemical properties indicate biological degradation of the polymer. The purpose of the present work was to compare the properties and biochemical degradation of soil humic acids from sod-podzol soil and chernozem, brown coal humic acid and melanins of soil fungi Aspergillus niger and Cladosporium cladosporiodes under conditions of a laboratory incubation experiment. The brown coal humic acid was selected as an inert sample, which had undergone both transformation during diagenesis and drastic chemical treatment during the industrial extraction from the coal, and which was expected therefore to be highly resistant to microbial degradation. The specific objectives of the work were: (1) to determine mineralization rate of humic acids and fungal melanins and (2) to study physico-chemical properties of humic acids and melanins and changes of these properties during 3 months of biodegradation.
2. Material and methods 2.1. Extraction of the humic acids and fungal melanins Humic acids were obtained by extraction with 0.1 M NaOH from air-dried soils ground to 1 mm. Humic acids were precipitated by acidification to pH 2 with HCl and purified by desalting with 0.4 M NaCl. For brown coal humic acid a commercial sodium humate, purified and transferred to H+-form, was used. The method used for culturing fungal mycelium was similar to that described by Linhares and Martin (1978). Briefly, the fungal cultures were grown in a mineral medium, containing (g l 1) 2.0 K2HPO4; 0.2 MgSO47H2O; 0.1 KCl; 2.0 NH4NO3; 30 glucose. Medium (200–250 ml) were placed in 900-ml Roux bottles and sterilized under pressure for 30min. The Roux bottles were incubated at 25 C on their flat sides until the pads became black from the melanin. After incubation the pads were removed from the culture medium, washed with distilled water and freeze-dried. Melanins were extracted from mycelium with 0.1 M NaOH, precipitated with HCl, purified and dried at 40 C. Ash content of all samples was <5% after purification. 2.2. Incubation experiments Samples of 100 mg of melanin or humic acid were thoroughly mixed with 20 g of quartz sand, 200 mg of
14
C-glucose was added, and the mixture was inoculated with 1 ml of aqueous extract containing an indigeneous microbial population of chernozemic soil. When mixed microbial population are used, the effect of individual soil microorganisms on organic matter decomposition is of minor importance (Dormaar, 1975). Glucose, considered as a carbon source easily decomposed by microorganisms, was added to stimulate microbial activity in the mixture and to prime decomposition of melanins and humic acids (Bingeman et al., 1953). Incubations were performed at 25 C in triplicate in 100 ml screw-capped jars, at 60% of field capacity. After 3 months of incubation melanins and humic acids were extracted from the mixture with 0.1 M NaOH and analyzed. Melanin and humic acid C lost by mineralization was the total amount of CO2-C evolved from the incubated mixture minus the amount of 14C-CO2 respired from decomposed 14C-glucose and 14C-labeled microbial tissues. The CO2 evolved was trapped in 8 ml 0.5 M KOH, placed in a plastic vial at the surface of the incubated mixture. To determine the amount of total CO2, at selected intervals, the vial was replaced from the jar and excess base was titrated with 1.0 M H2SO4 (Ivannikova, 1992). To determine the 14C-CO2 amount, 1 ml of KOH before titration was placed into a scintillator vial, 1 ml of scintillating mixture GS-13 was added and the radioactivity (count min 1) was measured on a LKB RackBeta 1217 liquid scintillation counter. Values of counts per minute were then converted into decays per minute using an internal standard method (L’Annunziata, 1979). 2.3. Analytical methods The C, H and N contents of initial and degraded humic acids and fungal melanins were determined using a Carlo-Erba 1106 CHNS elemental analyzer (T=1000 C). Functional groups in humic acids and melanins were determined by potentiometric titration. Preparations, diluted in 0.05 M NaOH (pH 11.7, concentration=0.5 mg/ml) were titrated with 0.1 M HCl at 25 C, ionic strength 0.1 (KCl) under permanent N2 flow. pH were recorded using a Horiba pH-meter after the value had remained stable for 1 min. To find the beginning and the end points of humic acids and melanins titration Gran’s analysis was used. Dissociation constants (pKa) were obtained as described by Takamatsu and Yoshida (1978). Infrared spectra were recorded on a Vector 22 spectrophotometer (Bruker, Germany) using KBr pellets, obtained by pressing, under vacuum, uniformly prepared mixtures of 1 mg dried preparation and 400 mg KBr. The UV and visible spectra of humic acids and melanins solutions were recorded at pH 11–12 on a Shimadzu
349
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
UV-260 spectrophotometer. The optical density (E4) was measured at wavelength 465nm, the extinctions, E 465 nm and E 665 nm, were used to calculate E4/E6 ratio. Molecular weight distributions of the melanins were obtained by gel filtration, using a Pharmacia column K 970 with Sephadex G-100 gel. Solution of 0.025 M Tris–HCl buffer (pH 8.1), 0.1% sodium dodecyl sulfate, 0.5 M NaCl and 0.02% NaN3 was used as an effluent. The elution rate was 3 ml cm 2 h 1. The optical density of the eluted solution was measured at wavelength 280 nm using a 2238 Uvicord S II detector (LKB Sweden). All samples were purified from low molecular weight contaminants by elution through a Sephadex G-10 column. Molecular weights of the fractions were calculated, using the formula developed by Determan (1971) for globular proteins, which was the following for Sephadex G-100 gel: log10M=5.941–0.847(Ve/V0), where M— molecular weight in kDa; Ve—elution volume in ml and V0—void volume in ml. Percentage content of each fraction was determined by calculating the area of the corresponding peak on the curve. The peak areas were calculated using ‘‘triangle method’’, developed for unresolved chromatographic peaks (Petzev and Kotzev, 1987). The distribution of hydrophobic and hydrophilic sites in humic acids and melanins was determined on a 1010 Amicon column, filled with octyl-sepharose CL4B gel. Elution was made at a rate of 10 ml cm 2 h 1 firstly with 0.05 M Tris–HCl buffer (pH 8.2) and then with 0.05 M Tris–HCl (pH 8.2)+0.3% Na-dodecylsulfate buffer using a step buffer change gradient. The step gradient was selected to divide the samples into two well-resolved fractions: fraction with predominance of hydrophilic properties (hydrophilic fraction) and fraction with predominance of hydrophobic properties (hydrophobic fraction). The content of the hydrophilic fraction was determined taking into consideration the corresponding peak area on the chromatogram.
(1982) for different soil systems. Subsequently mineralization of the unlabelled C from melanins and humic acids started, and for the next 10 weeks only 10% of the applied 14C evolved via decomposition and reutilization of 14C-labelled microbial tissues. After 91 days between 82 and 89% of the added 14C had been respired as CO2. All decomposition curves of the humic acids and fungal melanins follow an exponential pattern (Fig. 1). A relatively high mineralization rate for all samples was observed during the 1st month of incubation, after which the process decreased considerably. The decline of the mineralization process is known to be connected to the utilization of the labile part of melanins and humic acids and the formation of toxic products, that suppress microbial activity in the incubated mixture (Alexandrova, 1980). The decomposition rate of the humic acid from chernozem was 8% and for humic acid from sod-podzol soil—12% after 3 months of incubation. Coal humic acid was more resistant to biodegradation and showed only 3% loss of applied C for 3 months. Fungal melanins appeared to be more readily biodegradable: 22% C for A. niger and 25% C for C. cladosporiodes were released as CO2 during the experiment (Fig. 1). These values were comparable with those reported for some humic acids (Linhares and Martin, 1978; Alexandrova, 1980) and some melanins (Wolf and Martin, 1976; Boudot et al., 1989). The assessment of the incorporation of 14C from microbial biomass into humic acids and melanins (Table 1) showed that the highest level of incorporation was observed after 2 weeks of incubation (5–7% of applied 14C). After 3 months 14C was found only in sodpodzol humic acid and A. niger melanin. 3.2. Changes of physico-chemical properties of humic acids and fungal melanins during incubation Humic acid and melanin elemental composition, atomic ratios, functional group content and hydrophilic properties are shown in Table 2.
3. Results and discussion 3.1. Mineralization of humic acids and fungal melanins The CO2 emission from the incubated mixtures started on the 3rd day after inoculation. The lag-period observed was related to the slowing of microbial growth, caused by the high initial content of glucose untypical for usual soil conditions (Panikov et al., 1982). During the first two weeks of incubation mineralization of 14C-glucose and growth of 14C-labelled microbial biomass occurred. During this period about 70% of the applied 14C evolved as CO2. Similar mineralization rates for 14C-glucose (60–80% over 14 days) were reported by Cheshire et al. (1969), Dormaar (1975) and Zunino et al.
Table 1 Percentage of applied during incubation
14
C found in humic acids and melanins Days of uncubation
Sample
0
16
47
91
Sod-podzol humic acid Chernozem humic acid Brown coal humic acid Aspergillus niger melanin Cladosporium cladosporiodes melanin
– – – – –
6.1 5.0 4.5 7.0 5.0
1.9 – 1.2 3.1 –
2.2 – – 3.8 –
350
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Fig. 1. Mineralization rate of humic acids and fungal melanins (cumulative values). =confidence limits at 95% level.
Table 2 Analytical characteristics of humic acids and fungal melanins Sample
Content (at.%)
Atomic ratios
Functional groups
Content of hydrophilic
C
H
N
Oa
H/C
O/C
C/N
(mM( )/g)
fraction (%)
Sod-podzol humic acid
37.3b 36.5c
41.3 41.7
2.7 2.5
18.7 19.3
1.11 1.14
0.50 0.53
13.8 14.4
7.48 –
53 –
Chernozem humic acid
41.8b 39.0c
35.3 36.9
3.2 3.2
19.7 20.9
0.84 0.95
0.47 0.54
13.1 12.1
7.00 –
56 –
Brown coal humic acid
37.2b 39.2c
31.4 35.2
0.6 0.7
30.9 24.9
0.84 0.90
0.83 0.64
62.0 60.1
5.60 –
49 –
A. niger melanin
37.3b 38.2c
41.6 39.5
3.1 2.9
18.0 19.4
1.12 1.03
0.48 0.51
12.0 13.1
9.00 –
–
C.cladosporiodes melanin
33.9b 35.9c
45.2 39.7
4.0 4.5
16.9 20.0
1.33 1.11
0.50 0.58
8.5 8.1
10.56 –
14 –
a b c
By difference. Before incubation. After incubation.
Results of potentiometric titration of humic acids and fungal melanins in the pH region 4.0–10.5 show the presence of three types of functional groups: (1) with pKa value 4.4–4.7 (strong carboxylic groups, being titrated in pH range 3.5–7.0); (2) with pKa value 7.5–8.1 (weak carboxylic and phenolic, amine groups, being titrated in pH region 7.0–8.5); (3) with pKa value 9.6–9.9
(phenolic groups, being titrated in pH region over 8.5). Compared with the coal and soil humic acids, total content of functional groups of both fungal melanins was much higher, probably being evidence of higher reactivity of fungal melanins (Table 2). The infrared spectra of the melanins were found to be similar to those of the humic acids. Melanin from A.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
niger resembled IR-spectra of chernozem humic acid. IR-spectra of C. cladosporiodes melanin had some similarities with those of sod-podzol humic acid (Fig. 2). The elemental composition of the A. niger melanin was similar to those of the humic acids (Table 2). Melanin from C. cladosporiodes had a relatively low O content and high N content (4 at.%), compared with the soil and coal humic acids. The high N content was also reflected in the IR-spectrum of C. cladosporiodes melanin by an intense band at 1530 cm 1 (attributed to amide I protein linkages), indicating significant amounts of peptides. The band at 1530 cm 1 occurred with moderate intensity also in the spectrum of sod-podzol humic acid (Fig. 2). The H/C ratio, which is usually used to assess the contribution of aliphatic chains to the molecular structure, was the highest for the melanin from C. cladosporiodes and the bands at 2920–2860 cm 1 (aliphatic C– H stretching) were the most intense on the IR-spectrum of C. cladosporiodes melanin. According to the H/C ratio a high degree of aromaticity was obtained for the chernozem and brown coal humic acids (Table 2). This was confirmed by low absorption at 2920–2860 cm 1 and high absorption at 1610 cm 1, reflecting the abundant presence of C=C bonds, on the IR-spectra of both humic acids (Fig. 2). There were no substantial changes in the elemental composition of soil humic acids after 3 months of incubation, but a minor trend towards a decrease of C content and an increase of H content was observed
Fig. 2. Infrared spectra of humic acids and fungal melanins: (1) brown coal humic acid; (2) chernozem humic acid; (3) sodpodzol humic acid; (4) Cladosporium cladosporiodes melanin; (5) Aspergillus niger melanin.
351
(Table 2). This indicates, that slight loosening of the humic acid structure occurs due to microbial activity, and humic acid molecules become less condensed (Chernikov, 1992). The O content of the humic acid from the brown coal decreased after the experiment and the optical density of the humic acid decreased simultaneously (Table 3). This suggests a possible relationship between these two characteristics. For fungal melanins a decrease of H content and an increase of C content was observed after 3 months, that may be caused by an increase of the aromaticity of the pigments (Table 2). The results of hydrophobic chromatography are shown in Table 2. All humic acids consisted of two fractions—hydrophobic and hydrophilic—of almost equal abundances. Chernozem humic acid was the most hydrophilic. C. cladosporiodes melanin probably contained more hydrophobic sites than humic acids, based on the low content of hydrophilic fraction (Table 2). A. niger melanin sorbed on the octyl-sepharose gel, due to unspecific interactions. Raw precipitates of A. niger melanin are known to be highly hydrophilic, but lose hydrophilic properties after drying (Nicolaus, 1968). UV and visible absorption curves of humic acids were featureless (Fig. 3), with the exception of sod-podzol humic acid, which had a shoulder-like maximum in the 450 nm region and small maximum at 620 nm, reflecting the presence of Pg-pigment. Formation of such greencoloured pigments (so-called ‘‘Pg-fraction’’ of humic acids) in soil is due to the presence of certain fungi, such as Cenococcum graniforme, which synthesize a polynuclear quinoid pigment considered to be a perylene derivative (Sato and Kumada, 1967; Sato, 1976). Melanin from A. niger showed two broad, distinct maxima at 300 and 420 nm of unknown origin. The presence of such maxima may be connected to the contamination of the melanin with some non-melanin pigments, which contribute, according to Nicolaus (1968), up to 10% of the melanin’s weight. Spectral parameters (E4 extinction and E4/E6 ratio) of the fungal melanins (Table 3) were in the range reported for humic acids of different origin (Stevenson, 1982). However, the extinction coefficient of the initial melanin from C. cladosporiodes was much lower than for all humic acids studied and it was more similar to that of humic acids with a small degree of maturity and diagenesis (Chen et al., 1977). The E4-value of the A. niger melanin was the highest, but probably this value was overestimated due to the broad maximum at 420 nm. There were almost no changes in the optical properties of soil humic acids during incubation. At the same time changes of such properties for melanins were noteworthy (Table 3). After degradation the extinction of the A. niger melanin decreased, while the extinction of the degraded C. cladosporiodes melanin was three times greater than that of the initial pigment. At the same time the form of the UV-visible absorption curves of both
352
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Table 3 Changes of molecular weights and spectroscopic parameters of humic acids and fungal melanins during incubation Sample
Days of incubation
Spectroscopic parameters
Percentage of molecular weight fractions (kDa)
E4
E4/E6
>150
70
10
Sod-podzol humic acid
0 16 47 91
0.075 0.070 0.074 0.077
4.6 4.6 4.7 4.6
7 10 7 4
21 18 16 13
72 72 77 81
Chernozem humic acid
0 16 47 91
0.115 0.101 0.115 0.117
4.0 4.0 4.1 4.0
3 5 2 1
13 1 2 95
84 83 89 94
Brown coal humic acid
0 16 47 91
0.144 0.125 0.128 0.134
5.8 4.2 4.8 5.2
2 5 4 2
4 4 4 3
94 91 92 95
A. niger melanin
0 16 47 91
0.176 0.141 0.153 0.157
2.9 2.9 2.9 3.1
8 10 8 6
30 32 27 26
62 58 65 68
C. cladosporiodes melanin
0 16 47 91
0.030 0.041 0.080 0.096
3.6 3.6 3.7 3.7
40 42 29 22
42 44 24 28
18 14 47 50
Fig. 3. UV and visible spectra of humic acids and fungal melanins: (1) sod-podzol humic acid; (2) chernozem humic acid; (3) brown coal humic acid; (4) Aspergillus niger melanin; (5) Cladosporium cladosporiodes melanin.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
353
Fig. 4. Changes of molecular weight distributions of humic acids and fungal melanins during biodegradation.
melanins, and those of the humic acids, remained unchanged after 3 months of incubation. Also the E4/E6 ratios stayed almost constant for the duration of the experiment.
An increase of optical density together with an increase of O content of C. cladosporiodes melanin suggests that during degradation oxidizing reactions occur and that an additional amount of chromophoric groups
354
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
had formed in the melanin molecules. These O-containing chromophoric groups are probably formed by oxidation of the polypeptide or polysaccharide chains without cleavage of the aromatic rings. Such oxidative degradation reactions of the side chains in the melanin molecules result in a significant decrease in the molecular weight and in the simultaneous increase of the E4 value of C. cladosporiodes melanin. A. niger melanin is probably degraded by the destruction of the aliphatic chains with alternate double and single bonds, linked to the aromatic rings. The presence of these chains, linking aromatic rings, is considered to be one of the factors, which causes high optical density of humic acids (Orlov, 1990). The cleavage of the aliphatic chains with alternate double and single bonds reduces the extinction coefficient of A. niger melanin. The increase of the aromaticity of both pigments after incubation (Table 2) is also connected with the oxidative degradation of aliphatic components in the melanins. The molecular weight distributions of the initial melanins and humic acids were dissimilar (Fig. 4). Distribution curves of melanins had narrow, well-resolved peaks, that are typical for mixtures of individual components with high molecular weights. Similar distributions are obtained, for example, for mixtures of different proteins. In contrast humic acids, being polymolecular compounds, revealed a very different distribution of molecular sizes with relatively wide peaks. The molecular weight distributions of soil humic acids changed after incubation; the peaks became more symmetrical, indicating, that the fractions of these humic acids became less polydispersed. At the same time the molecular weight distributions of the melanins showed peaks widening (Fig. 4). On the gel-chromatographic curves of all tested samples three fractions were observed: (1) a maximum molecular weight above 150 kDa, (2) around 70 kDa and (3) around 10 kDa (Fig. 4). In Table 3 the percentage of each fraction is shown. The relative contents of the low molecular weight fraction (Mw 10 kDa) of the initial fungal pigments were lower than those found for the humic acid studied, especially of C. cladosporiodes. This finding is in accordance with the results obtained by Valmaseda et al. (1989) for melanins of fungal species, isolated from two Umbric Cambisols; molecular weights of those melanins were higher than for the humic acids of the corresponding soils. In our previous study melanins, obtained from 15 strains of soil fungi, were also found to have a relatively low content (10– 40%) of the fraction with Mw 10 kDa when compared to soil humic acids (Zavgorodnyaya et al., 2001). After 2 weeks of incubation, the high molecular weight fraction (Mw > 150 kDa) had increased for all polymers studied. These results, together with the high content of 14C in all polymers (Table 1), indicated that some organic compounds of microbial origin (large
fragments of peptides) had been incorporated into the humic acids and melanins after 2 weeks. After 6 weeks decrease of the high molecular weight fraction and of the 14C content suggests that these incorporated compounds had been utilized by microorganisms. After 3 months incubation the content of the low molecular weight fraction (Mw 10 kDa) increased and the content of the high molecular weight fractions decreased for all polymers with the exception of brown coal humic acid, which displayed insignificant changes (Table 3). For the incubated samples the changes of the initial distribution of the molecular weight fractions suggest different rates of fraction degradation. The high molecular weight fractions appeared to be less resistant to biodegradation and to form fragments with low molecular weight, which enlarged the fraction with Mw 10 kDa. The relative rate of degradation of the Mw 10 kDa fraction was lower, in comparison with the high molecular weight fractions, as a result accumulation of the low molecular weight fraction was observed for soil humic acids and fungal melanins. The most intense degradation was observed for the high molecular weight fractions of C. cladosporiodes melanin (Table 3). These fractions were probably enriched in the compounds (such as long peptide or carbohydrate chains), which were removed from the melanin molecule by microbial oxidative destruction.
4. Conclusion Some differences in functional groups content, spectral parameters and molecular weights between initial fungal melanins and humic acids were observed. However, for C. cladosporiodes melanin these differences were prominent, whereas A. niger melanin appeared to be more similar to the soil humic acids. During incubation fungal melanins were both intensively degraded by soil microorganisms, and after 3 months of degradation, became more like humic acids due to removal of aliphatic components, changes in elemental composition and in optical parameters and decrease of molecular weight. Humic acids under the same incubation conditions, even being partially mineralized, did not change their properties significantly. These results suggest that if melanin pigments of soil fungi, at least for these two species, are to become significant contributors to the stable soil organic fraction, they have to undergo a number of chemical transformations, drastically affecting the melanin properties. For further investigations of pigment contribution to humic acid accumulation, melanin studies of a large number of fungal species, along with quantitative assessment of annual melanin input to the soil, will be essential.
Y.A. Zavgorodnyaya et al. / Organic Geochemistry 33 (2002) 347–355
Acknowledgements The authors are grateful to the Russian Foundation for Basic Research (project Nos. 99-04-48007 and 00-1597856) and the Russian Federal Scientific Program (project 4.1.3) for support of this work. References Alexandrova, L.N., 1980. Soil Organic Matter and Processes of Its Transformation. Nauka, Leningrad. Bingeman, C.W., Varner, J.E., Martin, W.P., 1953. The effect of addition of organic materials on the decomposition of an organic soil. Soil Science Society of America Proceedings 17, 34–37. Boudot, J.-P., Bel Hadi Brahim, A., Steiman, R., Seigle-Murandi, F., 1989. Biodegradation of synthetic organo-metallic complexes of iron and aluminium with selected metal to carbon ratios. Soil Biology and Biochemistry 21, 961–966. Chen, Y., Senesi, N., Schnitzer, M., 1977. Information provided on humic substances by E4-to-E6 ratios. Soil Science Society of America Journal 41, 352–358. Chernikov, V.A., 1992. Transformation of humic acids as affected by autochtonous microflora. Russian Soil Science 3, 69–77. Cheshire, M.V., Mundie, C.M., Shepherd, H., 1969. Transformation of 14C glucose and starch in soil. Soil Biology and Biochemistry 1, 117–130. Determan, G., 1971. Gel-chromatography. Mir, Moscow. Dormaar, J.F., 1975. Susceptibility of organic matter of chernozemic Ah horizons to biological decomposition. Canadian Journal of Soil Science 55, 473–480. Ellis, D.H., Griffiths, D.A., 1974. The location and analysis of melanins in the cell walls of some soil fungi. Canadian Journal of Microbiology 20, 1379–1386. Filip, Z., Semotan, J., Kutilek, M., 1976. Thermal and spectrophotometric analysis of some fungal melanins and soil humic compounds. Geoderma 15, 131–142. Ivannikova, L.A., 1992. Using of absorption method for detecting of the natural CO2 efflux from a soil. Russian Soil Science 6, 133–139. Kang, K.S., Felbeck, G.T., 1965. A comparison of the alkaline extract of tissues of Aspergillus niger with humic acids from three soils. Soil Science 99, 175–181. Knicker, H., Almendros, G., Gonzalezvila, F.J., Ludemann, H.D., Martin, F., 1995. 13C and 15N NMR analysis of some fungal melanins in comparison with soil organic matter. Organic Geochemistry 23, 1023–1028. L’Annunziata, M.F., 1979. Radiotracers in Agricultural Chemistry. Academic Press, London.
355
Linhares, I.F., Martin, J.P., 1978. Decomposition in soil of the humic acid-type polymers (melanins) of Eurotium echinulatum, Aspergillus glaucus sp. and other fungi. Soil Science Society of America Journal 42, 738–743. Martin, J.P., Haider, K., 1969. Phenolic polymers of Stachybotrys atra, Stachybotrys chartarum and Epicoccum nigrum in relation to humic acid formation. Soil Science 107, 260–270. Nicolaus, R.A., 1968. Melanins. Hermann, Paris. Orlov, D.S., 1990. Humus Acids and the General Theory of Humification. MSU Press, Moscow. Panikov, N.S., Abu El-Naga, S.A., Zvyagintsev, D.G., 1982. Kinetics of glucose decomposition in soils. Soviet Soil Science 8, 70–77. Petzev, N., Kotzev, N., 1987. Manual on Gas Chromatography. Mir, Moscow. Sato, O, 1976. The chemical nature of components of Pg (green fraction of P-type humic acid). Soil Science and Plant Nutrition 22, 485–488. Sato, O., Kumada, K., 1967. The chemical nature of P-type humic acid. Soil Science and Plant Nutrition 13, 121–122. Stevenson, F.J., 1982. Humus Chemistry. Genesis, Composition, Reactions. Wiley and Sons, New York. Takamatsu, T., Yoshida, T., 1978. Determination of stability constants of metal-humic acid complexes by potentiometric titration and ion-selective electrodes. Soil Science 125, 377– 386. Valmaseda, M., Martinez, A.T., Almendros, G., 1989. Contribution by pigmented fungi to P-type humic acid formation in two forest soils. Soil Biology and Biochemistry 21, 23–28. Volnova, A.I., Mirchink, T.G., 1972. Fractionation of darkcolored fungi containing a green fraction. Soviet Soil Science 11, 140–145. Wolf, D.C., Martin, J.P., 1976. Decomposition of fungal micelia and humic-type polymers containing 14carbon from ring and side-chain labeled 2,4-D and chlorpropham. Soil Science Society of America Journal 40, 700–704. Zaprometova, K.M., Mirchink, T.G., Orlov, D.S., Yukhnin, A.A., 1971. Characteristics of black pigments of the darkcolored soil fungi. Soviet Soil Science 7, 22–30. Zavgorodnyaya, Yu.A., Demin, V.V., Kurakov, A.V., Biryukova, O.N., 2001. Characteristic of physico-chemical properties of fungal melanins. Moscow State University Herald, Series 17: Soil Science 3, 39–43. Zunino, H, Borie, F., Aguilera, S., Martin, J.P., Haider, K., 1982. Decomposition of 14C-labelled glucose, plant and microbial products and phenols in volcanic ash-derived soils of Chile. Soil Biology and Biochemistry 14, 37–43. Zviagintsev, D.G., Mirchink, 1986. On the nature of soil humic acids. Soviet Soil Science 5, 68–75.