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X-ray diffraction studies on the crystallinity and molecular weight of humic acids
Soil Biol. Biochem. Vol. 3, pp. 259-265. Pergamon Press 1971. Printed in Great Britain
X - R A Y D I F F R A C T I O N STUDIES ON THE CRYSTALLINITY A N D MOLECULAR WEIGHT OF HUMIC ACIDS S. A. VISSEP,and H. MENDEL Departments of Biochemistry and Chemistry, University of Ibadan, Ibadan, Nigeria
(Accepted 19 August 1970) Summary--Humic acids of different origins were investigated for crystallinity by means of Xray diffraction techniques. Only the humic acid formed in a culture of Aspergillusfiat,us on a modified Czapek-Dox medium which contained 0-1% phthalate was crystalline as found by electron microscopy and confirmed by X-ray diffraction techniques. Evidence showed that during the process of drying a rearrangement takes place in the humic acid which results in an ordered arrangement of the lattice structure. The nine lines of the powder diagram could be interpreted on the basis of a hexagonal unit cell with the dimensions a = 13"5/~ and c = 10-9 A_. On the strength of these findings a minimum molecular weight of M = 1393 was arrived at which is in good agreement with a series of molecular weights published for humic acids. By taking into account the effect of solvation a particle weight of 26,700 was calculated for humic acids suspended in water. INTRODUCTION THE first investigations of the structure of humic products by X-ray diffraction techniques
date back to 1935 when Sedletzky and Brunovsky reported a close structural relationship between lignins, humic acids, lignites, anthracites and graphite. Jodl (1941b, 1942) reported that humic acids extracted from Cassel brown coal consist of crystallites 8-18 A high and about 25 A~in diameter. Kasatochkin, Kukharenko, Zolotarevskaya and Razumova (1950) stated that X-ray diffraction patterns of humic acids that they extracted from a coal showed three maxima which they interpreted as corresponding to a double hexagonal carbon lattice similar to that in graphite. This interpretation of their data is, however, open to discussion. Riley (1949) proposed, without much experimental evidence, that the crystallites of humic acids from coal consisted of cross-linked aromatic nuclei, the linkage involving oxygen atoms and possibly also tilted benzene rings. With respect to the more recently formed compounds, resulting from an investigation of humic acids derived from peat, black earth formations and brown coal, Jung (1946) concluded that the acids themselves were not crystalline but could contain a slight amount of crystalline carbon of a graphite structure which could be embedded in " a n amorphousmesomorphous ground mass". G o r b u n o v (1947), who investigated humic acids from peat, found no signs of a crystalline structure in them. The diffuse X-ray diffraction rings which he sometimes obtained were interpreted as due to intra-molecular diffraction. D e b y e Scherrer rings were apparent only in samples containing more than 2 per cent of mineral salts. Neither could Flaig and Beutelspacher (1951), Nehring and Schiernann (1952), Kasatochkin (1953), K u m a d a (1956) or Visser (1963) establish with certainty the existence o f any crystallinity in their humic acid samples. They decided, therefore, that humic acids have a basically amorphous structure, and ascribed the occurrence of X-ray reflections to impurities such as clay particles, graphite and other crystalline or micro-crystalline matter. Thus, although humic acids extracted from highly carbonified materials such as coals 259
260
S.A. VISSER AND H. MENDEL
exhibit some crystalline features, the question is still open whether more recently formed humic products such as those found in various types of soils, litters, peats and microbial substrates also have a certain measure of crystallinity. There are, however, regularly recurring reports that the more recently formed humic acids occasionally show interference patterns which, according to the authors, are not due to mineral matter. Jodl (1941a) found, for instance, that whereas "humic acids" prepared from carbohydrates were entirely amorphous, phenol "humic acids" showed a certain degree of sub-microcrystallinity whilst, as already mentioned, some natural humic acids were found by him to be crystalline. In a later publication Jodl (1945) reported that both sodium humate and calcium humate had lattice spacings similar to the humic acids from which they were derived. Pallman (1942), from occasionally observed interference rings, attributed a weakly developed layer-lattice structure to relatively fresh humic material, whilst Riley (1949) also attributed a certain amount of crystallinity to it. Kasatochkin, Kononova, Larina and Egorova (1964) examined humic acid samples obtained from different soils and reported the presence of a (002) and ten other undefined diffraction bands indicating several types of inter-molecular regularity in these molecules. One was considered due to sheets of planar nuclei and another to the existence of side radicals. One of the reasons for the conflicting reports on the crystallinity of soil humic acids is probably the diffuse and heterogeneous nature of this material. This has become apparent from the many futile attempts which have so far been made to obtain these products in a pure form with well-defined physical and chemical constants. In order to investigate the existence of crystallinity in humic acids from a source where the mode of formation would give rise to the occurrence of products of similar origin, formed by a similar mechanism, humic material was extracted from a microbial culture which had been formed under well-defined chemical and physical conditions. The method of extraction could, under these circumstances, be a simple and gentle one so that it would be unlikely that artifacts would be formed. There would also be little opportunity for contamination by inorganic matter which could give rise to interference phenomena in the X-ray diffraction patterns. MATERIALS AND METHODS The microorganism selected for the production of humic matter was the fungus Aspergillusflavus Link which, when grown in Czapek-Dox medium has been shown to produce materials with properties identical to those of humic acids derived from soils and peats (Visser, 1968). The group of compounds investigated in this paper were obtained from substrates which had been incubated at 30°C for a period of 10 months. Because certain additives to Czapek-Dox medium had been found to stimulate the rate of humic acid production in previous experiments (Visser, 1970), the basic medium also invariably contained 0.5 ppm of manganese and 0-5 ppm of zinc. As sodium phthalate or sodium benzoate, when present at a concentration of 0-1 per cent had also been found to raise humic acid production, the fungus was grown on substrates containing one or the other of these substances (substrates I and II respectively). The humic acids were obtained by acidification with 1N sulphuric acid to pH 2.0 of the incubated and filtered substrate. The resulting precipitate was centrifuged off, repeatedly washed with distilled water until peptization started to occur and then dissolved at pH 8.0 using 1N sodium hydroxide. This process of acidification, washing and dissolution was repeated seven times, after which fractions of the humic acid preparation, in different states of dessication, were prepared for X-ray analysis. The elementary composition of the
X-RAY D I F F R A C T I O N STUDIES ON H U M I C ACIDS
261
acids is given in Table 1. Their i.r., u.v. and N M R spectra showed the presence of humic acids similar to those found in various types of soils (Visser, 1970). Apart from the solubility of its alkaline salts in water, the humic acid was also soluble in dimethylformamide, dioxan and pyridine. The product did not have a melting point as decomposition took place before this point was reached. TABLE 1. COMPOSITIONOF THE HUMICACIDSUSEDFOR X-RAY DIFFRACTION"STUDIES Medium
I (0" 1 ~ phthalate) II (0.1 ~ benzoate)
Ash content ( ~ )
C (~)
H (9~)
O (~)
N (~o)
< 0.03 <0"03
55- 44 52"47
6.99 5"91
30.07 35"87
7.50 5"75
In the case of the coloured lower molecular weight fractions (fulvic acids), the supernatant of the acidified substrate was neutralized to pH 7.0 with saturated barium hydroxide solution. The suspension was then centrifuged. Whilst the supernatant was kept for the lead acetate treatment (see following paragraph), the precipitate, after careful washing with distilled water, was treated with IN sulphuric acid until pH 2.0 was reached. Most of the coloured matter went into solution at this pH and it was separated from the insoluble barium sulphate by centrifugation. The supernatant was then extracted six times with nbutanol (1:3), the combined butanol extracts were neutralized with barium hydroxide, centrifuged, and then extracted with a small quantity of slightly alkaline water (pH 8.0). Next the water phase was acidified to pH 4.0 and extracted with some butanol until colourless. The butanol extract was then evaporated in a rotary film evaporator under reduced pressure, the residue taken up in absolute ethanol, centrifuged and the solvent evaporated. A brownish-yellow product resulted. A third fraction was obtained by adding an excess of a saturated solution of lead acetate to the neutral solution resulting from the barium hydroxide treatment. The resulting suspension was then centrifuged and treated in exactly the same way as described for the barium fraction. In this way all coloured matter was removed from the original substrate and three fractions were obtained varying in colour from brown (first fraction) to yellow (third fraction). These fractions were subjected to X-ray diffraction techniques after drying to varying degrees. RESULTS AND DISCUSSION
Powder photographs of humic acids, sealed in Lindemann capillaries, were taken with C u - I ~ radiation using the small Philips powder camera (diameter 57.54 mm). The diffraction patterns showed either a diffuse ring only, due to amorphous material, or a welldefined powder pattern, due to crystalline material and a weak diffuse ring. In the interpretation it was assumed that the diffuse rings originated from amorphous material only and that the powder pattern was due to crystalline material. It is then possible to interpret the powder pattern in terms of spacings by Bragg's law, viz. 2dhkl sin 0 = A and to interpret the diffuse rings according to Debye and Menke (James, 1948). Here the function sin sr/sr with s = 47r sin 0/A applies and only its first extreme value for sr = 1 "43~r was used, giving r ,~ 0.36 A/sin 0 where r is the most frequently occurring inter-atomic distance in the amorphous material.
262
s.A. VISSER AND H. MENDEL
X-ray photographs of moist, freshly precipitated humic acid from substrate I showed only two diffuse rings, the main one at r ~ 2 . 3 - 2 . 6 A and the weaker at r ~ 1 •5 A. The former value is probably an external diffraction effect due, most likely, to the water present, whereas the latter value is an internal diffraction effect, being the most frequently occurring intra-molecular distance. On air drying of the sample the main diffuse ring contracted, corresponding now to a distance of r ~ 3"0-3" 5 A, the subsidary diffuse ring disappeared and well-defined powder lines were observed. They are given in columns 2 and 3 of Table 2. The powder diagram could be interpreted on the basis of a hexagonal cell with a =- 13.9 A and c = 10-5 A. The indices based on this cell are given in column 1 and the calculated spacings, based on these indices and cell constants are given in column 4. As can be seen, reasonable agreement is obtained between calculated and observed spacings, their differences being < 1 per cent. On vacuum drying over phosphorous pentoxide, the material became well crystallized. Only one diffuse ring of very low intensity was present, corresponding to r ,~ 3" 1-3-5 A. The powder diagram could be interpreted on the basis of an hexagonal cell with a = 13-5 A and c ---- 10.9 A and a very good agreement between observed and calculated spacings was TABLE2.
OBSERVED AND CALCULATED CHARACTERISTICS OF CRYSTALLITES IN AIR-DRIED HUMIC ACIDS
Indices (hkil)
Intensity
dobs¢ rved
dealeulaled
2130, 3120, 32i0 3030, 3300 222~0, 4220 31211,4130, 43i0 2133, 3i23, 32i3 22213, 4223 42~0, 62210,62120 51~;0, 6150, 6~i0
Medium Medium Weak Strong Medium Weak Weak Strong
4- 53A 3"97A 3- 47~ 3" 16/~ 2" 78,& 2" 45A 2" 25,& 2.13A
4" 57A 4" 01d 3"47/~ 3" 17A 2" 77A 2" 46A 2.27A 2" 15A
obtained (see Table 3). As additional evidence of its hexagonal structure an electronmicrograph is shown of the humic acid obtained from substrate I. The presence is indicated o f hexagonal pencil-shaped crystals (Fig. 1). TABLE3. OBSERVED
AND CALCULATED CHARACTERISTICS OF CRYSTALLITES I N VACUUMDRIED HUMIC ACIDS
Indices (hkil)
Intensity
dobservea
dcatculat,d
1122 2iiO 3030 3300 2132 3122, 32i2 22210 4220 31211 4i31, 4311 22212 4222 31212 4i32, 43i2 33~063]0, 72130 5032 5302
Medium Medium Medium Medium Strong Weak Weak Weak Strong
4" 28/~ 3" 89A, 3"42A, 3- 38A 3" 12~ 2" 86A, 2" 76A 2" 25/~ 2" 15/~.
4" 27A, 3"89~ 3- 43A 3" 37A, 3" ll/~ 2- 87A 2.78A 2" 25A. 2' 15,~
FIG. 1. Humic acid crystals obtained from a fungal substrate.
SBB f.p. 2621
x 250,000.
X-RAY DIFFRACTION
STUDIES ON HUMIC ACIDS
263
The above results show clearly that on drying, humic acids can be obtained in a form giving X-ray patterns characteristic of crystalline material. It can be ruled out that the ensuring powder pattern is due to inorganic material, as the ash contents of the abovementioned samples were less than O-03 per cent (see Table 1). The water molecules initially present-possibly in a gel-like structure-are removed by drying, during which process a rearrangement takes place, resulting partly in an ordered arrangement and showing for the amorphous part an increase in value of inter-molecular distances (from N 2 *5 A to N 3 -2 A). The 2.5 A distance is probably due to water-water and water-humic acid inter-molecular distances, whereas the 3 *5A distance will be due mainly to inter-molecular distances between humic acid molecules. A similar phenomenon was observed with humic acids from substrate II. Here the main diffuse ring changed during drying from N 2 *3 A to N 3 -2 A. The subsidiary diffuse ring corresponding to N 1*5A remained constant during the process of drying. This substantiates the former deductions that the main diffuse ring is due to inter-molecular distances whereas the subsidiary one is caused by internal diffraction effects, being due to the humic acid only. The absence of any sharp diffraction lines indicated that these humic acids are amorphous. The humic acid fraction which was obtained by treatment of the substrate with barium hydroxide showed for the material obtained from either type of substrate only one diffuse ring at N 2.1 A. Faint diffraction lines present were due to barium sulphate which was present as a contaminant. The humic fraction precipitated with lead acetate gave powder patterns indicating the presence of crystalline material both for substrate I and II. In the case of the former the origin of these patterns could be traced to phthalic acid (lead phthalate being insoluble), whilst in the latter-a fraction made up of a brown-coloured polyunsaturated aliphatic carboxylic acid-we have not yet succeeded in tracing the origin of the crystalline features. As the ash content of this fraction was, however, relatively high, the results should be interpreted with care. The only humic fraction in which crystallinity has been established without any doubt is therefore the humic acid obtained from the incubated medium containing O-1 per cent phthalate. It appears that the presence of phthalate in the medium is essential for the occurrence of crystalline humic acid. The role of the phthalate in the formation of crystalline humic acid might indicate that the latter is some sort of derivative of the phthalates or of some metabolite formed from this material in the fungal substrate. The possibility that under the conditions of the experiment phthalate forms a crystalline chemical complex with humic acids is unlikely in view of the chemical properties of the humic acids. The formation of a crystalline structure because of physical interaction between humic acid and phthalate molecules would have been more likely if only several attempts of mild hydrolysis of the crystalline material had yielded some detectable amount of phthalate in solution. When humic acids obtained by the same extraction technique from the substrate of a much older fungal culture but grown on Czapek-Dox medium without any additives were investigated, no sign of crystallinity could be observed. As the weight of the unit cell equals the minimum molecular weight of a substance, this parameter M was calculated for the crystalline humic acid fraction according to the formula: M = p x N x V, where p represents the density of the investigated material (1.35 g/cm3), N is Avogadro’s number and V the volume of the asymmetric unit: a2c sin 120 A3 (1720 A”). Consequently a value of 1392 was found as the weight of the humic acid represented as (C64H97026N&, with II = 1. This value is in reasonable agreement with a series of molecu-
264
S. A. VISSER AND H. MENDEL
lar weight determinations on humic acids of which the average value is approximately 1300 (see Table 4). As we estimated that as a result of solvation the volume of a humic acid molecule in a water phase increased approximately 25 times, it was found that the particle weight of humic acids in water would be approximately 26,700 or a multiple of this, assuming mice1 formation. These values are of the same magnitude as the majority of the values TABLE4. VALUESOF ‘MOLECULAR WEIGHTS’REPORTEDFORHUMICACIDS Value
Method of estimation
-1000 984-1294 1336 1350 1235-1445 1684
Isothermic distillation End-group analysis Equivalent weight Freezing point
500@-7000 Dialysis Diffusion 45’00-26,000 14,000-20,000 Gel filtration Gel filtration 5000-100,000 (average 25,000) 10,00&200,000 Sedimentation, - 25,000 Viscosity - 36,000 Osmometry 47,000-53,800 - 53,000 Sedimentation
Reference
Welte et al. (1954) Fuchs (1930) Oden (1919) Arnold et al. (1934) Samec and Pirkmaier (1930) Schnitzer and Desjardins (1962)
Flaig and Beutelspacher (1954) Scheele (1937) Bailly ahd Margulis (1968) Mehta ef al. (1963) viscosity
Robert-G&o et al. (1966) Piret et al. (1960) Visser (1964) Wright et al. (1958) Stevenson et al. (1953)
of the second series found by methods based on viscosity, rate of sedimentation, rate of dialysis and gel filtration. These results seem to substantiate the possibility that the minimum values of the unit cell as indicated before (a = 13.5 A; c = IO.9 A) are, in fact, the real values. Acknowledgement-We are grateful to Mr D. SANNI for his assistance diffraction photographs.
in the preparation
of the X-ray
REFERENCES ARNOLDC. L., LOWYA. and THIE~SENR. (1934) Isolation and Study of the Humic Acids from Peat. Report of Investigation, U.S. Bureau of Mines, No. 3258. BAILLYJ. R. and MARGULISH. (1968) Etude de quelques acides humiques sur gel de dextrane. PI. Soil. 29, 343-361. FLAIG W. and BEUTELSPACHER H. (1951) Zur Kenntnis der Huminsaure. II. Elektronenmikroskopische Untersuchungen an nattirlichen und synthetischen Huminsaure. Z. PflErniihr. Dung. Bodenk. 52, 1-21. FLAIG W. and BEUTEL~PACHER H. (1954) Physikalische Chemie der Huminslure. Landbouwk. Tijdschr., ‘s-Grav. 66,306335. FUCHSW. (1930) Uber die Entstehung der Kohlen nach dem gegenwlrtigen Stand der chemischen Forschung. Brennst.-Chem. 11, 106-112. GORBUNOVN. I. (1947) X-ray and electronographic studies of humic acid, humic substances and humates. Pedoiogy 1947,240-248. JAMESR. W. (1948) The Crystalline State-II. The Optical Principles of the Difiaction of X-rays, Bell, London. JODL R. (1941a) Rontgenographic researches on natural and artificial humic substances. Brennst.-Chem. 22, 157-161. JODL R. (1941b) Size and shape of humic acid crystallites. Brennst.-Chem. 22,256257. JODL R. (1942) The relationship between hymatomelanic acids and humic acids. Brennst.-Chem. 23,259-263. JODL R. (1945) Rontgenographische Untersuchungen tiber Natrium-humate. Bodenk. PfiErntihr. 35,222-228.
X-RAY DIFFRACTION JUNG E. (1946) Uber den Feinbau
STUDIES ON HUMIC ACIDS
265
der Humusstoffe. Z. PflErniihr. Dung. Bodenk. 37,2-16. KASATOCHKIN V. I. (1953) The structure of carbonized substances. Izvest. Akad. Nauk SSSR Otdel. Tekh. Nauk 1401-1416. KASATOCHKINV. I., KONONOVAM. M., LARINAN. K. and EGOROVA0. I. (1964) Theses on ‘Spectral and X-ray Investigations of Chemical Structure of Humic Substances of Soil’, Transactions of the Eighth Znternational Congress of Soil Science, pp. 81-86. KASATOCHKIN V. I., KUKHARENKOT. A., ZOLOTAREVSKAYA E. Yu. and RAZUMOVAL. L. (1950) X-ray study of humic acids of coal. Dokl. Akad. Nauk SSSR 74,775-178. KUMADAK. (1956) Physico-chemical studies on humic acid formation. IX. X-ray diffraction analysis and coagulation by electrolytes. J. Sci. Soil Manure, Tokyo 27,21; 79-82. MEHTAN. C., DUBACHP. and DEUELH. (1963) Untersuchungen iiber die Molekulargewichtsverteilung von Huminstoffen durch Gelfiltration an Sephadex. Z. PflEmiihr. Dung. Bodenk. 102, 128-137. NEHRINGK. and SCHIEMANNR. (1952) Untersuchungen zum Humusproblem. I. Beitrage zur Kenntnis der Vorglnge bei der Rotte von Stallmist und Komposten sowie zur Kenntnis der Huminslure. II. Beitrlge zur Kenntnis der Huminslure. Z. PfErniihr. D&g. Bodenk. 57, 193-215. ODEN S. (1919) Die Huminsauren. Kolloidchem. Beih. 11. 75-260. PALLMANNH. (1942) Dispersoidchemische Probleme in der Humusforschung. Koiloidzeitschrift 101, 72-81. PIRET E. L., WHITE R. G., WALTHERH. C., and MADDENA. J. (1960) Some physico-chemical properties of peat humic acids. Scient. Proc. R. Dubl. Sot. Ser. Al, 69-79. RILEYH. L. (1949) An X-ray diffraction study of humification. J. Soil Sci. 1,104111. ROBERT-GEROM., HARDISSONC., LE BORGNEL. and PIGNAUDG. (1966) Etude biochimique des humus et des substances para-humiques de synthese microbienne en fonction de leur poids mol&culaire. Annfs Inst. Pasteur 111, 750-767. SAMECM. and PIRKMAIERB. (1930) Zur Kenntnis der Huminsauren. Kolloidzeitschrift 51,96-100. SCHEELEW. (1937) Beitrlge zur Charakterisierung natiirlicher Humusstoffe. Kolloidchem. Beih. 46,368-424. SCHNITZERM. and DESJARDINSJ. G. (1962) Molecular and equivalent weights of the organic matter of a podzol. Proc. Soil Sci. Sot. Am. 26, 362-365. SEDLETZKYI. D. and BRUNOVSKYB. K. (1935) Structure of humic acid and its relation to Ii&n and coal. Kolloidzeitschrifr 73,9&91. . ’ STEVENSON F. J., VANWINKLEQ. and MARTINW. P. (1953) Physico-chemical investigations of clay-absorbed organic colloids-IT. Proc. Soil Sci. Sot. Am. 17,31-34. VISSERS. A. (1963) Electron-microscopic and electron-diffraction patterns of humic acids. Soil Sci. 96, 353356. VISSERS. A. (1964) A physico-chemical study of the properties of humic acids and their changes during humification. J. Soil Sci. 15,202-219. VINIERS. A. (1968) Studies on Aspergillusfkzvus Link and its metabolites. V. The production of polyphenolic compounds. W. Afr. J. biol. appl. Chem. 10,31-41. VI~SERS. A. (1970) Studies on Aspergillusflavus Link and its metabolites. VII. Investigation of the optimum conditions for the formation of humic compounds in Aspergillusflavus cultures, and some properties of the products formed. W. Afr. J. biol. appl. Chem. 13,3-13. WELTE E., NEUMANNA. and ZIECHMANNV. (1954) Uber Huminsluren. II. Molekular-und Aquivalentgewicht. Naturwissenschaften 41,334. WRIGHTJ. R., SCHNITZERM. and LAVICKR. (1958) Some characteristics of the organic matter extracted by dilute inorganic acids from a podzolic B-horizon. Can. J. Soil Sci. 38, 14-22.
X - R A Y D I F F R A C T I O N STUDIES ON THE CRYSTALLINITY A N D MOLECULAR WEIGHT OF HUMIC ACIDS S. A. VISSEP,and H. MENDEL Departments of Biochemistry and Chemistry, University of Ibadan, Ibadan, Nigeria
(Accepted 19 August 1970) Summary--Humic acids of different origins were investigated for crystallinity by means of Xray diffraction techniques. Only the humic acid formed in a culture of Aspergillusfiat,us on a modified Czapek-Dox medium which contained 0-1% phthalate was crystalline as found by electron microscopy and confirmed by X-ray diffraction techniques. Evidence showed that during the process of drying a rearrangement takes place in the humic acid which results in an ordered arrangement of the lattice structure. The nine lines of the powder diagram could be interpreted on the basis of a hexagonal unit cell with the dimensions a = 13"5/~ and c = 10-9 A_. On the strength of these findings a minimum molecular weight of M = 1393 was arrived at which is in good agreement with a series of molecular weights published for humic acids. By taking into account the effect of solvation a particle weight of 26,700 was calculated for humic acids suspended in water. INTRODUCTION THE first investigations of the structure of humic products by X-ray diffraction techniques
date back to 1935 when Sedletzky and Brunovsky reported a close structural relationship between lignins, humic acids, lignites, anthracites and graphite. Jodl (1941b, 1942) reported that humic acids extracted from Cassel brown coal consist of crystallites 8-18 A high and about 25 A~in diameter. Kasatochkin, Kukharenko, Zolotarevskaya and Razumova (1950) stated that X-ray diffraction patterns of humic acids that they extracted from a coal showed three maxima which they interpreted as corresponding to a double hexagonal carbon lattice similar to that in graphite. This interpretation of their data is, however, open to discussion. Riley (1949) proposed, without much experimental evidence, that the crystallites of humic acids from coal consisted of cross-linked aromatic nuclei, the linkage involving oxygen atoms and possibly also tilted benzene rings. With respect to the more recently formed compounds, resulting from an investigation of humic acids derived from peat, black earth formations and brown coal, Jung (1946) concluded that the acids themselves were not crystalline but could contain a slight amount of crystalline carbon of a graphite structure which could be embedded in " a n amorphousmesomorphous ground mass". G o r b u n o v (1947), who investigated humic acids from peat, found no signs of a crystalline structure in them. The diffuse X-ray diffraction rings which he sometimes obtained were interpreted as due to intra-molecular diffraction. D e b y e Scherrer rings were apparent only in samples containing more than 2 per cent of mineral salts. Neither could Flaig and Beutelspacher (1951), Nehring and Schiernann (1952), Kasatochkin (1953), K u m a d a (1956) or Visser (1963) establish with certainty the existence o f any crystallinity in their humic acid samples. They decided, therefore, that humic acids have a basically amorphous structure, and ascribed the occurrence of X-ray reflections to impurities such as clay particles, graphite and other crystalline or micro-crystalline matter. Thus, although humic acids extracted from highly carbonified materials such as coals 259
260
S.A. VISSER AND H. MENDEL
exhibit some crystalline features, the question is still open whether more recently formed humic products such as those found in various types of soils, litters, peats and microbial substrates also have a certain measure of crystallinity. There are, however, regularly recurring reports that the more recently formed humic acids occasionally show interference patterns which, according to the authors, are not due to mineral matter. Jodl (1941a) found, for instance, that whereas "humic acids" prepared from carbohydrates were entirely amorphous, phenol "humic acids" showed a certain degree of sub-microcrystallinity whilst, as already mentioned, some natural humic acids were found by him to be crystalline. In a later publication Jodl (1945) reported that both sodium humate and calcium humate had lattice spacings similar to the humic acids from which they were derived. Pallman (1942), from occasionally observed interference rings, attributed a weakly developed layer-lattice structure to relatively fresh humic material, whilst Riley (1949) also attributed a certain amount of crystallinity to it. Kasatochkin, Kononova, Larina and Egorova (1964) examined humic acid samples obtained from different soils and reported the presence of a (002) and ten other undefined diffraction bands indicating several types of inter-molecular regularity in these molecules. One was considered due to sheets of planar nuclei and another to the existence of side radicals. One of the reasons for the conflicting reports on the crystallinity of soil humic acids is probably the diffuse and heterogeneous nature of this material. This has become apparent from the many futile attempts which have so far been made to obtain these products in a pure form with well-defined physical and chemical constants. In order to investigate the existence of crystallinity in humic acids from a source where the mode of formation would give rise to the occurrence of products of similar origin, formed by a similar mechanism, humic material was extracted from a microbial culture which had been formed under well-defined chemical and physical conditions. The method of extraction could, under these circumstances, be a simple and gentle one so that it would be unlikely that artifacts would be formed. There would also be little opportunity for contamination by inorganic matter which could give rise to interference phenomena in the X-ray diffraction patterns. MATERIALS AND METHODS The microorganism selected for the production of humic matter was the fungus Aspergillusflavus Link which, when grown in Czapek-Dox medium has been shown to produce materials with properties identical to those of humic acids derived from soils and peats (Visser, 1968). The group of compounds investigated in this paper were obtained from substrates which had been incubated at 30°C for a period of 10 months. Because certain additives to Czapek-Dox medium had been found to stimulate the rate of humic acid production in previous experiments (Visser, 1970), the basic medium also invariably contained 0.5 ppm of manganese and 0-5 ppm of zinc. As sodium phthalate or sodium benzoate, when present at a concentration of 0-1 per cent had also been found to raise humic acid production, the fungus was grown on substrates containing one or the other of these substances (substrates I and II respectively). The humic acids were obtained by acidification with 1N sulphuric acid to pH 2.0 of the incubated and filtered substrate. The resulting precipitate was centrifuged off, repeatedly washed with distilled water until peptization started to occur and then dissolved at pH 8.0 using 1N sodium hydroxide. This process of acidification, washing and dissolution was repeated seven times, after which fractions of the humic acid preparation, in different states of dessication, were prepared for X-ray analysis. The elementary composition of the
X-RAY D I F F R A C T I O N STUDIES ON H U M I C ACIDS
261
acids is given in Table 1. Their i.r., u.v. and N M R spectra showed the presence of humic acids similar to those found in various types of soils (Visser, 1970). Apart from the solubility of its alkaline salts in water, the humic acid was also soluble in dimethylformamide, dioxan and pyridine. The product did not have a melting point as decomposition took place before this point was reached. TABLE 1. COMPOSITIONOF THE HUMICACIDSUSEDFOR X-RAY DIFFRACTION"STUDIES Medium
I (0" 1 ~ phthalate) II (0.1 ~ benzoate)
Ash content ( ~ )
C (~)
H (9~)
O (~)
N (~o)
< 0.03 <0"03
55- 44 52"47
6.99 5"91
30.07 35"87
7.50 5"75
In the case of the coloured lower molecular weight fractions (fulvic acids), the supernatant of the acidified substrate was neutralized to pH 7.0 with saturated barium hydroxide solution. The suspension was then centrifuged. Whilst the supernatant was kept for the lead acetate treatment (see following paragraph), the precipitate, after careful washing with distilled water, was treated with IN sulphuric acid until pH 2.0 was reached. Most of the coloured matter went into solution at this pH and it was separated from the insoluble barium sulphate by centrifugation. The supernatant was then extracted six times with nbutanol (1:3), the combined butanol extracts were neutralized with barium hydroxide, centrifuged, and then extracted with a small quantity of slightly alkaline water (pH 8.0). Next the water phase was acidified to pH 4.0 and extracted with some butanol until colourless. The butanol extract was then evaporated in a rotary film evaporator under reduced pressure, the residue taken up in absolute ethanol, centrifuged and the solvent evaporated. A brownish-yellow product resulted. A third fraction was obtained by adding an excess of a saturated solution of lead acetate to the neutral solution resulting from the barium hydroxide treatment. The resulting suspension was then centrifuged and treated in exactly the same way as described for the barium fraction. In this way all coloured matter was removed from the original substrate and three fractions were obtained varying in colour from brown (first fraction) to yellow (third fraction). These fractions were subjected to X-ray diffraction techniques after drying to varying degrees. RESULTS AND DISCUSSION
Powder photographs of humic acids, sealed in Lindemann capillaries, were taken with C u - I ~ radiation using the small Philips powder camera (diameter 57.54 mm). The diffraction patterns showed either a diffuse ring only, due to amorphous material, or a welldefined powder pattern, due to crystalline material and a weak diffuse ring. In the interpretation it was assumed that the diffuse rings originated from amorphous material only and that the powder pattern was due to crystalline material. It is then possible to interpret the powder pattern in terms of spacings by Bragg's law, viz. 2dhkl sin 0 = A and to interpret the diffuse rings according to Debye and Menke (James, 1948). Here the function sin sr/sr with s = 47r sin 0/A applies and only its first extreme value for sr = 1 "43~r was used, giving r ,~ 0.36 A/sin 0 where r is the most frequently occurring inter-atomic distance in the amorphous material.
262
s.A. VISSER AND H. MENDEL
X-ray photographs of moist, freshly precipitated humic acid from substrate I showed only two diffuse rings, the main one at r ~ 2 . 3 - 2 . 6 A and the weaker at r ~ 1 •5 A. The former value is probably an external diffraction effect due, most likely, to the water present, whereas the latter value is an internal diffraction effect, being the most frequently occurring intra-molecular distance. On air drying of the sample the main diffuse ring contracted, corresponding now to a distance of r ~ 3"0-3" 5 A, the subsidary diffuse ring disappeared and well-defined powder lines were observed. They are given in columns 2 and 3 of Table 2. The powder diagram could be interpreted on the basis of a hexagonal cell with a =- 13.9 A and c = 10-5 A. The indices based on this cell are given in column 1 and the calculated spacings, based on these indices and cell constants are given in column 4. As can be seen, reasonable agreement is obtained between calculated and observed spacings, their differences being < 1 per cent. On vacuum drying over phosphorous pentoxide, the material became well crystallized. Only one diffuse ring of very low intensity was present, corresponding to r ,~ 3" 1-3-5 A. The powder diagram could be interpreted on the basis of an hexagonal cell with a = 13-5 A and c ---- 10.9 A and a very good agreement between observed and calculated spacings was TABLE2.
OBSERVED AND CALCULATED CHARACTERISTICS OF CRYSTALLITES IN AIR-DRIED HUMIC ACIDS
Indices (hkil)
Intensity
dobs¢ rved
dealeulaled
2130, 3120, 32i0 3030, 3300 222~0, 4220 31211,4130, 43i0 2133, 3i23, 32i3 22213, 4223 42~0, 62210,62120 51~;0, 6150, 6~i0
Medium Medium Weak Strong Medium Weak Weak Strong
4- 53A 3"97A 3- 47~ 3" 16/~ 2" 78,& 2" 45A 2" 25,& 2.13A
4" 57A 4" 01d 3"47/~ 3" 17A 2" 77A 2" 46A 2.27A 2" 15A
obtained (see Table 3). As additional evidence of its hexagonal structure an electronmicrograph is shown of the humic acid obtained from substrate I. The presence is indicated o f hexagonal pencil-shaped crystals (Fig. 1). TABLE3. OBSERVED
AND CALCULATED CHARACTERISTICS OF CRYSTALLITES I N VACUUMDRIED HUMIC ACIDS
Indices (hkil)
Intensity
dobservea
dcatculat,d
1122 2iiO 3030 3300 2132 3122, 32i2 22210 4220 31211 4i31, 4311 22212 4222 31212 4i32, 43i2 33~063]0, 72130 5032 5302
Medium Medium Medium Medium Strong Weak Weak Weak Strong
4" 28/~ 3" 89A, 3"42A, 3- 38A 3" 12~ 2" 86A, 2" 76A 2" 25/~ 2" 15/~.
4" 27A, 3"89~ 3- 43A 3" 37A, 3" ll/~ 2- 87A 2.78A 2" 25A. 2' 15,~
FIG. 1. Humic acid crystals obtained from a fungal substrate.
SBB f.p. 2621
x 250,000.
X-RAY DIFFRACTION
STUDIES ON HUMIC ACIDS
263
The above results show clearly that on drying, humic acids can be obtained in a form giving X-ray patterns characteristic of crystalline material. It can be ruled out that the ensuring powder pattern is due to inorganic material, as the ash contents of the abovementioned samples were less than O-03 per cent (see Table 1). The water molecules initially present-possibly in a gel-like structure-are removed by drying, during which process a rearrangement takes place, resulting partly in an ordered arrangement and showing for the amorphous part an increase in value of inter-molecular distances (from N 2 *5 A to N 3 -2 A). The 2.5 A distance is probably due to water-water and water-humic acid inter-molecular distances, whereas the 3 *5A distance will be due mainly to inter-molecular distances between humic acid molecules. A similar phenomenon was observed with humic acids from substrate II. Here the main diffuse ring changed during drying from N 2 *3 A to N 3 -2 A. The subsidiary diffuse ring corresponding to N 1*5A remained constant during the process of drying. This substantiates the former deductions that the main diffuse ring is due to inter-molecular distances whereas the subsidiary one is caused by internal diffraction effects, being due to the humic acid only. The absence of any sharp diffraction lines indicated that these humic acids are amorphous. The humic acid fraction which was obtained by treatment of the substrate with barium hydroxide showed for the material obtained from either type of substrate only one diffuse ring at N 2.1 A. Faint diffraction lines present were due to barium sulphate which was present as a contaminant. The humic fraction precipitated with lead acetate gave powder patterns indicating the presence of crystalline material both for substrate I and II. In the case of the former the origin of these patterns could be traced to phthalic acid (lead phthalate being insoluble), whilst in the latter-a fraction made up of a brown-coloured polyunsaturated aliphatic carboxylic acid-we have not yet succeeded in tracing the origin of the crystalline features. As the ash content of this fraction was, however, relatively high, the results should be interpreted with care. The only humic fraction in which crystallinity has been established without any doubt is therefore the humic acid obtained from the incubated medium containing O-1 per cent phthalate. It appears that the presence of phthalate in the medium is essential for the occurrence of crystalline humic acid. The role of the phthalate in the formation of crystalline humic acid might indicate that the latter is some sort of derivative of the phthalates or of some metabolite formed from this material in the fungal substrate. The possibility that under the conditions of the experiment phthalate forms a crystalline chemical complex with humic acids is unlikely in view of the chemical properties of the humic acids. The formation of a crystalline structure because of physical interaction between humic acid and phthalate molecules would have been more likely if only several attempts of mild hydrolysis of the crystalline material had yielded some detectable amount of phthalate in solution. When humic acids obtained by the same extraction technique from the substrate of a much older fungal culture but grown on Czapek-Dox medium without any additives were investigated, no sign of crystallinity could be observed. As the weight of the unit cell equals the minimum molecular weight of a substance, this parameter M was calculated for the crystalline humic acid fraction according to the formula: M = p x N x V, where p represents the density of the investigated material (1.35 g/cm3), N is Avogadro’s number and V the volume of the asymmetric unit: a2c sin 120 A3 (1720 A”). Consequently a value of 1392 was found as the weight of the humic acid represented as (C64H97026N&, with II = 1. This value is in reasonable agreement with a series of molecu-
264
S. A. VISSER AND H. MENDEL
lar weight determinations on humic acids of which the average value is approximately 1300 (see Table 4). As we estimated that as a result of solvation the volume of a humic acid molecule in a water phase increased approximately 25 times, it was found that the particle weight of humic acids in water would be approximately 26,700 or a multiple of this, assuming mice1 formation. These values are of the same magnitude as the majority of the values TABLE4. VALUESOF ‘MOLECULAR WEIGHTS’REPORTEDFORHUMICACIDS Value
Method of estimation
-1000 984-1294 1336 1350 1235-1445 1684
Isothermic distillation End-group analysis Equivalent weight Freezing point
500@-7000 Dialysis Diffusion 45’00-26,000 14,000-20,000 Gel filtration Gel filtration 5000-100,000 (average 25,000) 10,00&200,000 Sedimentation, - 25,000 Viscosity - 36,000 Osmometry 47,000-53,800 - 53,000 Sedimentation
Reference
Welte et al. (1954) Fuchs (1930) Oden (1919) Arnold et al. (1934) Samec and Pirkmaier (1930) Schnitzer and Desjardins (1962)
Flaig and Beutelspacher (1954) Scheele (1937) Bailly ahd Margulis (1968) Mehta ef al. (1963) viscosity
Robert-G&o et al. (1966) Piret et al. (1960) Visser (1964) Wright et al. (1958) Stevenson et al. (1953)
of the second series found by methods based on viscosity, rate of sedimentation, rate of dialysis and gel filtration. These results seem to substantiate the possibility that the minimum values of the unit cell as indicated before (a = 13.5 A; c = IO.9 A) are, in fact, the real values. Acknowledgement-We are grateful to Mr D. SANNI for his assistance diffraction photographs.
in the preparation
of the X-ray
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