- Email: [email protected]
Preparation and characterization of model humic polymers containing organic phosphorus
Soil Biol. Biochem.Vol. 17, No. 2, pp. 213-219, 1985 Printed in Great Britain. All rights reserved
0038.0717/8553.00+ 0.00 Copyright Q 1985Pergamon Press Ltd
PREPARATION AND CHARACTERIZATION OF MODEL HUMIC POLYMERS CONTAINING ORGANIC PHOSPHORUS CHRISTINE A. BRANNON and LEE E. SOMMERS Department of Agronomy, Purdue University, West Lafayette, IN 47907, U.S.A.
Summary-The nature of organic P in soil organic matter was studied by evaluating the incorporation of serine, phosphoserine, ethanolamine, phosphoethanolamine and glycerophosphate into model humic polymers prepared by chemical oxidation of polyphenols. Elemental and functional group analysis indicated that the composition of model humic polymers ranged as follows: organic C, .50.6-56.8%; total acidity, 7.86-l 1.87 m-equiv g-r; carboxyl, 1.42-2.00 m-equiv g-i; total hydroxyl, 6.79-10.0 m-equiv g-‘; ash, 6.4-13.9°/0; E,/E, ratio, 5.34-6.19; organic N, [email protected]/~ and organic P, 0.254-&9420/~.These values are within the ranges reported for soil humic substances. The onIy non-phenoiic compounds incorporated into model humic polymers were those containing free amino groups. The P content of model polymers was not increased by the presence of KH,PO,, glycerophosphate, serine or ethanolamine whereas phosphoserine and phosphoethanolamine resulted in model polymers containing 0.254 and 0.942% P, respectively. Further characterization studies of the model polymer containing phosphoethanolamine (HA-PE) showed that most of the C (83.27$, N (79.8%) and P (75.3%) was in the humic acid fraction. Gel filtration of HA-PE showed that 0.5% of the polymer was present in high molecular weight (mol. wt) components (mol. wt > l~,O~) and 74.8% of the polymer was in two components of mol. wt lO,OOO-50,000.The majority of the organic Pin HA-PE was associated with the medium molecular weight fractions (79.2%) while 16.8% of the P was associated with materials possessing mol. wt < 10,000. Attempts to demonstrate the presence of organic P functional groups contained in HA-PE by infrared spectroscopy was limited by the relatively small amounts of organic P incorporated into the model humic polymers. The results obtained show that a portion of the unidentified organic P in soil humic substances may arise from the inco~oration of organic compounds wntaining both amino and phosphate ester functional groups during oxidative ~lymerization of polyphenols.
INTRODUCTION
The chemical nature of soil organic P has not been fully elucidated although several forms of organic P have been identified including inositol-P esters, phospholipids and nucleic acids. Various studies indicate that <60x of soil organic P is present as known biochemicals such as inositol phosphates (Anderson, 1980). Organic P has been detected in the humic fraction of soils, especially in high molecular weight fractions (Dormaar, 1972; Moyer and Thomas, 1970; Swift and Posner, 1972; Thomas and Bowman, 1966). Typically, >40% of the total organic P in soils is associated with the fulvic and humic fractions. This association warrants investigation as a probable source for some of the uncharacterized organic P in soil. Nitrogenous compounds containing free -NH, groups have been covalently linked, via nucleophilic addition, to aromatic rings of synthetic humic polymers (Haider et al., 1965; Flaig, 1966; Ladd and Butler, 1966a, b; Haider and Martin, 1967, 1970; Martin and Haider, 1969; Bondietti et ai., 1972). Phospho~lated organic compounds containing a free -NH2 group may also be incorporated into humic substances through a mechanism similar to that suggested for the incorporation of organic N. Organic P compounds incorporated during the formation of humic molecules would be an integral part of the humic structure.
Our objective was to demonstrate that organic P compounds can be covaientiy Linked into model humic polymers prepared by oxidative polymerization of poiyphenois. The characteristics of organic P in the model humic polymers were evaluated by elemental, functional group, solubility, gel filtration and infrared spectroscopic analyses.
213
MATERIALS AND METHODS
Preparation
of model
humic polymers
Model humic polymers were synthesized in the presence and absence of low molecular weight N or P compounds by chemical oxidation of a mixture of aromatic compounds (Table 1). To insure that organic P was covalently bonded to humic polymers, contamination of polymers with P was minimized by eliminating phosphate buffers and by using 0, as the oxidant in the absence of mushroom tyrosinase, which was found to contain P. Phosphate present in either a buffer or tyrosinase could become adsorbed during the synthesis of humic polymers, resulting in overestimation of organic P contained in the model polymers. Therefore, model humic polymers were prepared by chemical rather than enzymatic oxidation. For each polymer, 3 mmol of each of the phenolic compounds listed in Table 1 were added to 2.5 1 of deionized H,O and the solution adjusted to pH 7 with 6 N NaOH to ensure dissolution of all
214
CHRISTINE A.
BRANNON and LEEE. SOMMERS
Table 1. Aromatic compounds used to synthesize model humic polymers Toluenes 2,3-Dihydroxytoluene 2,6-DihydroxytoIuene 3,4-Dihydroxytoloene 3,SDihydroxytoluene
(4-homopyr~at~hoi) (orcinol)
Cinnamic acids o-Coumaric m-Coumaric p-Coumaric 3,4-Dihydroxy~innami~ acid (caffeic) 4-Hydroxy,3-methoxycinn~ic acid (ferulic) Benzenes phenols 1,3-Dihydroxybenzene (resorcinol) 1,2,3-Ttihydroxybenzeae (pyrogallol) 1,3,5-Ttihydroxybenzene (phloroglucinol) Benzoic acids o-Hydroxybenzoic acid (salicyclic) ~-Hydroxy~nzoic acid p-Hydroxybenzoic acid 2,3-Dihydroxybenzoic acid (o-pyrccatechuic) 2,4-Dihydroxybenzoic acid (/?-resorcyclic) 2,5-Dihydroxybenzoic acid (gentistic) 2.6-Dihvdroxvbenzoic acid fy-resorcvclic) 3:CDih;drox&enzoic acid (~rotocakchtkc) 3,5-Dihydroxybenzoic acid (a-resorcyclic) 2,4,6-T~hydroxy~nzoic acid 3,4,5-T~hydroxy~nzo~c acid (gallicf 4-Hydroxy,3-methoxybenzoic acid (vanillic) 4-Hydroxy,3,5-dimethoxybenzoic acid (syringic) Amino compounds Swine (HA-S) Ethanolamine (HA-E) Phosphate compounds KWQ, Cilycerophosphate (HA-GP) Phosuhoserine (HA-P9 Phosbboethanoiamine (HA-PE) “Abbreviation
for synthetic polymer prepared.
reagents. The desired amino or phosphate compound (18 mmol) was then added to the mixture of polyphenols. The solution was adjusted to pH 8.5 with 3 N NaOH and aerated for 7 days at room temperature (22”C), by which time the solution had turned dark brown or black. The suspension of synthetic humic material was concentrated to approximately 500ml in a forced-air oven (7o”C), thoroughly dialyzed against frequent changes of deionized H,O and concentrated to a small volume at 70°C. The humic polymers were freeze-dried and stored at room temperature in the dark. The abbreviations used for each model humic polymer are shown in Table 1 and they will be used throughout this section. The control polymer was prepared by oxidizing the aromatic compounds without any N or P amendments. Characterization
qf model humic polymers
Total C content was determined by wet oxidation (Nelson and Sommers, 1974), total N by the microKjeldahl method (Nelson and Sommers, 1972) and total P by calorimetry (Murphy and Riley, 1962) after perchloric acid digestion (Sommers and Nelson, 1972). The total N and P content was also determined for the N and P compounds used to evaluate the purity of the reagents used. Total acidity was determined by the Ba(OH), method of Schnitzer and Gupta (1965). Carboxyl content was estimated by the modified calcium acetate method of Woltzclaw and
Sposito (1979). Total hydroxyl content was taken as the difference between total acidity and carboxyl content. Moisture and ash content were determined gravimet~c~ly after drying at 105°C for 15 h and heating in a mufIle furnace at 650°C for 4 h, respectively. The Ed/E6 ratios were determined by dissolving 0.5 mg of model humic polymer in 25 ml of 0.05 N NaHC03 and measuring the absorbance at 465 and 665 nm on a Beckman Model 24 spectrophotometer. Infrared spectroscopy (IR) was used to determine the presence of functional groups in model humic polymers containing organic P. Pressed KBr discs were prepared by mixing 0.5 mg of phosphoethanolamine (PE), HA-E or HA-PE with 300 mg of ground KBr in a mortar and pestle, transferring the mixture to a KBr die and applying 455 MPa of pressure under a vacuum for 15 min. The IR spectra were obtained with a Perkin-Elmer Model 180 double-beam grating spectrometer. The percent transmittance and corresponding wavenumber (4000-250 cm-‘) were recorded for each OScm-’ interval and stored on cassette tapes. Before plotting with a Houston Instruments X-Y plotter, all IR spectra were smoothed using a 25 point mathematical routine. A blank KBr disc was placed in the reference beam for all analyses. The following references were used in making infrared band assignments: Bellamy (1975); Sadtler SIPS (1967); White (1964); Alpert et al. (1970); Filip et al. (1974); Stevenson and Goh (1971). The distribution of organic P in model humic polymers was determined by physical and chemical fractionation. The model humic polymer HA-PE was fractionated by gel filtration to estimate molecular weight and P distribution. A sample of humic acid (acid-insoluble, alkali-soluble) extracted from a Chalmers silt loam soil (fine-silty, mixed, mesic, Typic Haplaquoll) was also analyzed by gel filtration. Sephadex G- 100 (Pharmacia Fine Chemicals, Uppsala, Sweden) was poured into a glass column (2.5 x 100 cm). Tris (2-amino-2(hydroxymethyl)propane- 1,3-diol) buffer (50.14 g Tris f 4.2 ml cont. HCl 1-l at pH 9) was used as the eluant, as suggested by Swift and Posner (1971) to overcome the gel-solute interactions frequently observed in gel filtration of humic materials. Humic acid samples (10 mg) were dissolved in 1 ml Tris and loaded onto the column, Effluent was collected (flow rate of 42 ml h-*) and the absorbance of each 1Oml fraction was measured at 410nm and an aliquot was analyzed for total P following perchloric acid digestion (Sommers and Nelson, 1972). The amount of P was obtained by referring to a standard curve (O-5 ,ug P) prepared from a standard KH,PO, solution. The Sephadex G-100 column was standardized with Blue Dextran 2000 (Pharmacia Fine Chemicals) and methyl organge (mol. wt 327). The amount of Blue Dextran and methyl orange contained in each fraction was determined at 610 and 510 nm, respectively using a Beckman Model 24 spectrophotometer. The mode1 humic polymer HA-PE was fractionated into the humic acid (acid insoluble, alkali soluble) and fulvic acid (acid soluble, alkali soluble) components. Freeze-dried HA-PE (approx. 0.3 g) was dissolved in 5 ml of 0.5 N NaOH, adjusted to pH 1 with cont. HCl, equilibrated for 16 h and centri-
215
Preparation of model humics containing organic P Table 2. Elemental and functional group analysis of model humic polymers” Total Compound added during humic synthesi@ None KH,PO, Glycerophosphate Serine Phosphoserine Ethanolamine Phosphoethanolamine
Apparent yield’ (g) 4.2 4.0 2.7 3.3 4.4 3.6 5.6
&I% ratio
Ash
5.36 -6.19 5.51 5.34 5.54 5.57 5.59
6.97 9.81 9.22 12.69 11.23 6.37 13.90
.~
N -~
--F%) $4.8 55.3 53.5 55.6 53.8 50.6 56.8
P
0.03 0.04 0.09 0.70 0.20 1.65 0.56
--.
0.006 0.002 0.016 0.002 0.254 0.001 0.942
Total acidity p( 10.46 9.88 10.68 8.70 7.86 8.62 11.87
Total Carboxyld hydroxyP’ m-equiv g-I)------~__ 1.56 8.90 1.60 8.22 1.73 8.95 2.00 6.79 1.47 7.86 I.60 6.89 1.77 10.00
*All values reported on a moisture and ash-free weight basis. bRefer to Table I for listing of compounds used to synthesize model humic polymers. ‘g product recovered following lyophilization. dMethod of Holtzclaw and Sposito (1979). ‘Total hydroxyl content determined by difference (total acidity--COOH).
fuged. The humic acid precipitation process was repeated and the final solid material rinsed with deionized H,O. The supernatants from all the centrifugation were pooled to yield the fulvic acid fraction, Subsamples of the HA-PE model polymer and the precipitated humic acid were then analyzed for total C, N and P. The C, N and P contents of the fulvic acid fraction were determined by difference between the original polymer and the humic acid fraction. RESULTS
Characterization of model humic polymers The chemical oxidation of polyphenolic mixtures in the presence and absence of P-containing compounds yielded between 2.7 and 5.6 g humic polymer (Table 2). The greatest quantity of humic polymer was formed in the presence of compounds containing both amino and phosphate groups, i.e. 4.4g for HA-PS and 5.6 g for HA-PE. Decreased yield of humic polymers was found for systems amended with serine and ethanolamine (3.3-3.6 g). Humic polymers prepared in the absence of organic P or N compounds (control and KH,PO,) yielded similar amounts of product (-4 g) while the least amount of model humic polymer was synthesized in the presence of glycerophosphate (2.7 g). The organic C content of the model humic polymers ranged from 50.6 (HA-E) to 56.8% C (HA-PE) and averaged 54.3% C. Model humic polymers synthesized in the presence of non-nitrogenous compounds (control, KH2P04 and HA-GP) contained no organic N. The organic N content of humic polymers prepared in the presence of non-phosphorylated N compounds was somewhat lower than expected, ranging from 0.70 (HA-S) to 1.65% N (HA-E). A comparison of the N content in polymer HA-PE vs HA-E and polymer HA-PS vs HA-S indicated that the presence of phosphorylated analogues decreased the N content of the synthetic humic polymers by 66.1 and 71.4x, respectively. Model humic polymers synthesized in the presence of KH,PO.,, glycerophosphate, serine and ethanolamine contained no organic P. The humic polymers prepared in the presence of phosphorylated N compounds contained 0.254 and 0.942% P for HA-PS and HA-PE, respectively. Total acidity of the model humic polymers ranged from 7.86 to 11.87 m-equiv g-‘. Carboxyl contents ranged from 1.47 to 2.00 m-equiv g-’ while hydroxyl contents ranged from 6.79 to 10.00 m-
equiv g-‘. The ash content of the model humic polymers was somewhat higher than expected, ranging from 6.4 (HA-E) to 13.9% (HA-PE). The preparations were not acidified prior to dialysis and thus Na + was likely associated with the functional groups. As expected, the E,/E6 ratio was similar for all model polymers ranging from 5.34 to 6.19, indicating similar molecular complexity of the polymers. The N or P content of the model polymers along with yield data enable calculation of the proportion of added N or P incorporated into the polymers. Greater proportions of ethanolamine were incorporated into model humic polymers than serine as reflected by 17.5 and 5.7% of the added N being found in HA-E and HA-S, respectively. In comparison, only 5.6 and 1.3% of the added organic P was incorporated into HA-PE and HA-PS, respectively. It should also be noted that the molar ratio of N/P in HA-PS and HA-PE is greater than the 1:1 ratio of the PS and PE added to systems for synthesis of model humics indicating that there was some hydrolysis of phosphate esters during the in~~oration of PS and PE into model humics. The overall similarity in major IR absorption bands of synthetic humic polymers with and without PE was expected since they were both synthesized from the same polyphenolic precursors (Fig, 1). A striking feature of both spectra is the strong and very broad absorbance band centered around 3400 cm-‘. This band arises from the H-bonded OH stretching mode arising from COOH, phenol&OH and polymeric intermolecular H-bonded OH groups. The infrared spectra of the model humic polymers formed through the chemical oxidation and subsequent polymerization of polysubstituted phenolic subunits resemble previously published spectra of model phenolic polymers (Bondietti et al., 1972; Filip et d., 1974; Mathur and Schnitzer, 1978; Wang et al., 1978); fungal polymers (Filip er al., 1974) and soil humic substances (Schnitzer and Gupta, 1964; Stevenson and Goh, 1971; Schnitzer and Khan, 1972; Filip et al., 1974; Tan, 1976; Eltantawy and Baverez, 1978). The aromatic C-N stretching vibration characteristic of secondary aromatic amines at 1275 cm-’ indicates the presence of N in the model humic polymers. Although secondary aromatic amine N-H stretching vibrations occur in the vicinity of the 1275 cm-’ band (Bellamy, 1975), they are generally overlaid by the stronger aromatic C-N
CHRISTINE
2750 75
1 2125
Wovenumber
1500
I .9+5
A. BRANNON and LEE E. SOMMERS
250
(cm-‘)
Fig. 1. Infrared spectra of model humic polymers. A, 0.5 mg of HA-E polymer and B, l.Omg of HA-PE polymer. absorbances. The C-O stretching vibrations and OH deformations of COOH may also account for the 1275 cm-’ band since they characteristically absorb between 1320 and 1211 cm-‘. Assignments for the major infrared absorption bands of the model humic polymers are summarized in Table 3. Organic P incorporat:d into model humic polymers as PE was examined with IR. The infrared spectra were recorded of (1) PE; (2) model humic polymer containing PE (HA-PE) and; (3) for humic polymer containing E (HA-E). A sharp well-defined spectrum was obtained for PE while diffuse, broad peaks were obtained for both model humic polymers. The absorption bands corresponding to organic P functional groups of PE were: P=O, free (1256 cm-‘); P=O, H-bonded (1168 cm-’ and P-O-C (1185, 1032, 945 and 770cm-‘). The P-OH absorbantes were broad, shallow bands between 2560 and
2700cm-’ (Bellamy, 1975) and are of little use in identifying phosphorylated compounds. Infrared absorption bands characteristic of organic P functional groups were not detectable in the spectrum of HA-PE. Gel chromatography confirmed the polydisperse nature of model humic substances. The continuous elution pattern exhibited by both the model humic polymer (HA-PE) and the soil humic acid indicated that molecular weights ranged from ~2500 to > 100,000. Gel filtration of HA-PE indicated the presence of three major mol. wt species: a component with mol. wt > 100,000 and two medium mol. wt components (15,000 and 20,000 (Fig. 2)). Based on absorbance at 410nm, most of the HA-PE humic polymer was distributed in components II and III. Comparatively little of the HA-PE polymer was found in fraction I. The distribution of molecular weight components differed for synthetic and soil humic substances (Table 4). The soil humic acid contained greater amounts (16.0%) of high mol. wt (> 50,000) constituents compared with the model humic polymer (2.3%). The model humic polymer was composed predominantly of medium mol. wt (lO,OOO-50,000) components (74.8%) as compared to the soil humic acid (37.8%). The soil humic acid contained a larger proportion of low mol. wt (< 10,000) constituents (46.7%) than the model humic polymer (22.9%). The distribution of P for HA-PE and soil HA also differed considerably (Fig. 2). Most of the P contained in HA-PE (79.2%) was associated with the medium mol. wt (1O,OOO-50,000)fractions while very little P (3.9%) was detected in the high mol. wt (> 50,000) fractions (Table 4). In contrast, the distribution of P contained in the soil HA was shifted towards the higher mol. wt (>50,000) fractions (39.7%) with a considerable amount of P (15.90/,) associated with the > 100,000 mol. wt fraction. Interestingly, although a sizeable proportion of the soil HA (46.7%) was found in low mol. wt (-c 10,000) fractions, only trace amounts of the P were associated with these fractions. In contrast, a similar proportion of the total polymer and total P was associated with low mol. wt fractions (22.9 and 16.83/ respectively)
Table 3. The main IR absorption bands of the model humic polymers HA-E and HA-PE Frequency (cm-‘) .__ 3400 2925
Assignment
Intensity’
Polymeric OH stretch, H-bonded vs, broad C-H stretch of benzene and w to m phenolic-CH, 2925, 2860 Aliphatic C-H stretch of CH, w to m 1700 C=O stretch of aryl acids m 1612 Aromatic skeletal ring breathing YS mode; C=C conjugated to C=O; COO1515 Aromatic skeletal ring breathing mode m 1455 Aromatic skeletal ring breathing mode; sh aliphatic C-H deformation of CH, 1387 C-O stretch and OH deformation of S phenolic-OH; COO1275 C-N stretch of secondary aromatic amine; m N-H stretch of secondary aromatic amine; C-O stretch and OH deformation of COOH 1167 C-O stretch and OH deformation of COOH w 1080 Aromatic C-O stretch and OH deformations w 1030 (0-CH,?) w 800 Aromatic C-H out-of-plane deformations m ‘vs, very strong; s, strong; m, medium; w, weak; sh, shoulder.
217
Preparation of model humics containing organic P 0.5 c
r
10
25,000
Void volume
15
20
25
30
35
10.000
40
45
Fraction
50
55
60
65
70
75
00
number
Fig. 2. Fractionation of HA-PE (-) and humic acid isolated from Chalmers soil (---) on Sephadex G-100. Humics dissolved in 1 ml of 0.41 M Tris were applied to 2.5 x 100cm column of Sephadex G-100 and eluted with 0.41 M Tris. Ten ml fractions were collected at a flow rate of 42 ml h-’ and analyzed for P and absorbance at 410 nm. of HA-PE. About 99% of the P applied to the Sephadex G-100 column was recovered indicating that HA-PE was not adsorbed onto the gel matrix in the presence of the Tris buffer eluant. The P content of the soil HA was estimated to be 0.87% P which favorably compares with the 0.73% P content of HA-PE. The model humic polymer HA-PE was separated into humic and fulvic acid fractions on the basis of solubility in acid. The majority of the C (83.2x), N (79.8%) and P (75.3%) originally contained in HA-PE was found in the acid insoluble humic acid fraction. The molar N/P ratios of HA-PE and the resulting humic acid indicated that the C-O-P bonds in HA-PE were stable to chemical hydrolysis during the humic-fulvic acid fractionation procedure.
DISCUSSION
Infrared spectroscopy was not particularly informative in the detection of organic P functional groups in the model humic polymers. Either the concentrations of organic P were below the detection limits of the infrared techniques employed or the absorbantes normally assignable to organic P were obscured by stronger or broad absorptions attributed to other functional groups in the model humic polymers. Two major elution bands corresponding to high mol. wt and low mol. wt components were found
for model humic substances. The elution pattern observed in the gel filtration of the soil humic acid agrees with the gel chromatography studies of soil humic substances of Moyer and Thomas (1970), Swift and Posner (1971) and Tan (1976). The bimodal elution pattern observed from the gel filtration of the HA-PE model humic polymer yielded mol. wt fractions which differed from the anticipated mol. wt distribution characteristic of soil humic substances. The dominance of medium mol. wt (10,00~50,000) constituents and the relative absence of high mol. wt (> 50,000) components could be expected during the synthesis of HA-PE because simple polyphenols underwent chemical oxidation and subsequent polymerization into humic substances. Within one week, the phenols had been transformed into low (< 10,000) and medium (lO,OOO-50,000) mol. wt polycondensates. Only a small amount of high mol. wt (> 50,000) material would be expected to be formed within this period, especially in the absence of polymerizing enzymes (e.g. phenolase) coupled with the non-availability of larger organic compounds (e.g. proteins, carbohydrates) commonly found in plant or animal residues. The relative absence of high mol. wt constituents in synthetic humic polymers compared with soil humic acids was reported in the gel filtration studies of Ladd and Butler (1966b) and Haider and Martin (1970). Haider and Martin (1970) demonstrated that high mol. wt fractions of synthetic phenolic humic polymers could be increased up to 6%
Table 4. The molecular weight and phosphorus distribution in synthetic humic containing phosphoethanolamine (HA-PE) and soil humic acid as determined by fractionation on Seohadex G-100 Percent of weight Molecular weight fraction > 100,000 50,00&100,000 25,OOC-50,000 10,00&25,000 < 10,000
HA-PE 0.5 1.8 22.1 52.1 22.9
Soil humic acid 1.3
a.1 9.5 27.8 46.1
Percent of phosphorus HA-PE
Soil humic acid
1.1 2.8 21.4 57.8 16.8
15.9 23.8 38.5 21.8 trace
218
CHRISTINEA. BRANNONand LEE E. SOMMERS
through the incorporation of nitrogenous compounds such as glycine, peptone or albumin into the polymers. Model humic polymers synthesized by fungi grown on a variety of media exhibited a mol. wt distribution similar to soil humic acids (Ladd and Butler, 1966b; Martin et al., 1979; Martin and Haider, 1969; Haider and Martin, 1970). Organic P has frequently been reported in association with soil humic substances, in particular with high mol. wt fractions. The majority of organic P contained in HA-PE polymer (75.3%) was found in the humic acid fraction. Fractionation by gel filtration of the soil humic acid indicated that approximately 40% of the P was associated with high mol. wt (>50,000) components. Moyer and Thomas (1970) reported a similar distribution of organic P with 36% contained in high mol. wt (> 50,000) fractions while Thomas and Bowman (1966) observed somewhat higher proportions of organic P (74.5%) in the high mol. wt (> 50,000) fractions of humic acid. Swift and Posner (1972) also found that the majority of P in humic acids extracted from Australian soils was in association with high mol. wt fractions. About SOT/, of the P contained in the model HA-PE humic polymer was associated with medium (lO,OOO-50,000) mol. wt fractions. The distribution of Pin the various mol. wt fractions of humic substances would be expected to shift towards higher mol. wt fractions with time. Similar trends in the mol. wt distribution of both N and P in humic acids were reported by Swift and Posner (1972), supporting the mechanism advanced earlier that organic P may be incorporated into humic substances through the interaction of the free amino groups of a phosphorylated nitrogenous compound with phenolic subunits. Thus, during polymerization, organic P is first associated with low and medium mol. wt humic components. With time, the organic P becomes associated with higher mol. wt fragments as the phenolic constituents continue to condense and incorporate other available organic compounds. Easily-degradable organic P would be microbially decomposed during the early stages of humus formation. The resulting highly-resistant organic P fraction associated with high mol. wt components of humic acid would remain essentially non-available to both plants and microorganism. In summary, this study demonstrated that phosphate esters can be incorporated into humic materials if the organic P compound contains a free amino group. Martin, Haider and others (Haider et al., 1965; Flaig, 1966; Ladd and Butler, 1966a, b; Haider and Martin, 1967, 1970; Martin and Haider, 1969; Bondietti et al., 1972) have shown that compounds containing -NH, functional groups undergo nucleophilic addition to activated phenolic compounds. Thus, one mechanism by which organic P may become associated with the soil humic fraction involves incorporation of phosphorylated organic compounds group during oxidative containing a free -NH, polymerization of polyphenols. Organic P compounds thus incorporated would become an integral part of the soil humic material and this mechanism may explain the character of a portion of the currently undefined organic P associated with soil orgamc matter.
REFERENCES Alpert N. L., Keiser W. E. and Szymanski H. A. (1970) Theory and Practice of Infrared Spectroscopy.
Plenum
Press, New York. Anderson G. (1980) Assessing organic phosphorus in soils. In The Role of Phosphorus in- Agri<u~e (F. E. Kha-
sawneh. E. C. SamDle and E. J. Kammath. Eds).
PP.
411-43i. American’ Society of Agronomy, Ma&s&, Wisconsin. Bellamy L. J. (1975) The Infrared Spectra of Complex Molecules. Wiley, New York. Bondietti E., Martin J. P. and Haider K. (1972) Stabilization of amino sugar units in humic type polymers. Soil Science Society of America Proceedings 36, 591-602.
Dormaar J. F. (1972) Chemical properties of organic matter extracts from a number of A, horizons by a number of methods. Canadian Journal of Soil sciences 52, 67-71. Filip Z., Haider K., Beutelspacher H. and Martin J. P. (1974) Comparisons of IR-spectra from melanins of microscopic soil fungi, humic acids and model phenol polymers. Geoderma 11, 31-52. Flalg W. (1966) The chemistry of humic substances. In The Use of Isotopes in Soil Organic Matter Studies, pp. 103-127. Pergamon Press, Oxford. Haider K. and Martin J. P. (1967) Synthesis and transformation of phenolic compounds by Epicoccum nigrum in relation to humic acid formation. Soil Science Society of America Proceedings 31, 766-712.
Haider K. and Martin J. P. (1970) Humic acid-type phenolic polymers from Aspergillus sydowi culture medium, Stathybofrys spp. cells and autoxidized phenol mixtures. Soil Biology & Biochemistry 2, 145-156.
Haider K., Frederick L. R. and Flaig W. (1965) Reactions between amino acid compounds and phenols during oxidation. Plant and Soil 22, 49-64. Holtzclaw K. M. and Sposito G. (1979) Analvtical properties of the soluble, metal-complkxing’ fractions in sludgesoil mixtures: IV. Determination of carboxyl groups in fulvic acid. Soil Science Society of America Journal 43, 3 18-323.
Ladd J. N. and Butler J. H. A. (1966a) Comparison of properties of synthetic and natural humic acids. In The Use of Isotopes in Soil Organic Maiter Studies, pp. 143-159. Pergamon Press, Oxford. Ladd J. N. and Butler J. H. A. (1966b) Comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating amino derivatives. Australian Journal of Soil Research 4, 41-54.
Martin J. P. and Haider K. (1969) Phenolic polymers of Stachybotrys atra, Stachybotrys chartarum and Epieoccum nigrum in relation to humic acid formation. Soil Science 107, 260-270.
Martin J. P., Haider K. and Linhares L. F. (1979) Decomposition and stabilization of ring “‘C-labeled catechol in soil. Soil Science Society of American Journal43, 10&104. Mathur S. P. and Schnitzer M. (1978) A chemical and spectroscopic chracterization of some synthetic analogues of humic acids. Soil Science Societv_ of” America Journal 42, 591-596.
Moyer J. R. and Thomas R. L. (1970) Organic phosphorus and inositol phosphates in molecular size fractions of a soil organic matter extract. Soil Science Society of America Proceedings 34, 80-83.
Murphy J. and Riley J. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31-36. Nelson D. W. and Sommers L. E. (1972) A simple procedure for estimation of total nitrogen in soils and sediments. Journal of Environmental Quality 1, 423425.
Nelson D. W. and Sommers L. E. (1974) A rapid and accurate procedure for estimation of organic carbon in soils. Proceedings of Indiana Academy of Sciences 84, 456-462.
Preparation of model humics containing organic P Sadtler Standard Infrared Prism Spectra (1976) Sadtler Research Laboratories, Philadelphia. Schnitzer M. and Gupta U. C. (1964) Some chemical characteristics of the organic matter extracted from the 0 and B2 horizons of a gray wooded soil. Soil Science Society
of America
Proceedings
28, 374-377.
Schnitzer M. and Gupta U. C. (1965) Determination acidity in soil organic matter. Soil Science Society America
Proceedings
of
29, 272-277.
36, 902.-904.
Stevenson F. J. and Goh K. M. (1971) Infrared spectra of humic acids and related substances. Geochemica et Cosmochimica
Acta 35, 47 l-483.
Swift R. S. and Posner A. M. (1971) Gel chromatography of humic acid. Journal of Soil Science 22, 237-249. Swift R. S. and Posner A. M. (1972) Nitrogen, phosphorus and sulphur contents of humic acids fractionated with respect to molecular weight. Journal of Soil Science 23, SO-57.
of
Schnitzer M. and Khan S. U. (1972) Humic Substances in the Environment. Dekker, New York. Sommers L. E. and Nelson D. W. (1972) Determination of total phosphorus in soils: A rapid perchloric acid digestion procedure. Soil Science Society of America Proceedings
219
Tan K. H. (1976) Complex formation between humic acid and clays as revealed by gel filtration and infrared spectroscopy. Soil Biology & Biochemistry 8, 235-239. Thomas R. L. and Bowman B. T. (1966) The occurrence of high molecular weight organic phosphorus compounds in soil. Soil Science Society of America Proceedings 30, 799-80 1.
Wang T. S. C., Li S. W. and Huang P. M. (1978) Catalytic polymerization of phenolic compounds by a latosol. Soil Science 126, 81-87. White R. G. (1964) Handbook of Industrial Infrared Analysis. Plenum Press, New York.
0038.0717/8553.00+ 0.00 Copyright Q 1985Pergamon Press Ltd
PREPARATION AND CHARACTERIZATION OF MODEL HUMIC POLYMERS CONTAINING ORGANIC PHOSPHORUS CHRISTINE A. BRANNON and LEE E. SOMMERS Department of Agronomy, Purdue University, West Lafayette, IN 47907, U.S.A.
Summary-The nature of organic P in soil organic matter was studied by evaluating the incorporation of serine, phosphoserine, ethanolamine, phosphoethanolamine and glycerophosphate into model humic polymers prepared by chemical oxidation of polyphenols. Elemental and functional group analysis indicated that the composition of model humic polymers ranged as follows: organic C, .50.6-56.8%; total acidity, 7.86-l 1.87 m-equiv g-r; carboxyl, 1.42-2.00 m-equiv g-i; total hydroxyl, 6.79-10.0 m-equiv g-‘; ash, 6.4-13.9°/0; E,/E, ratio, 5.34-6.19; organic N, [email protected]/~ and organic P, 0.254-&9420/~.These values are within the ranges reported for soil humic substances. The onIy non-phenoiic compounds incorporated into model humic polymers were those containing free amino groups. The P content of model polymers was not increased by the presence of KH,PO,, glycerophosphate, serine or ethanolamine whereas phosphoserine and phosphoethanolamine resulted in model polymers containing 0.254 and 0.942% P, respectively. Further characterization studies of the model polymer containing phosphoethanolamine (HA-PE) showed that most of the C (83.27$, N (79.8%) and P (75.3%) was in the humic acid fraction. Gel filtration of HA-PE showed that 0.5% of the polymer was present in high molecular weight (mol. wt) components (mol. wt > l~,O~) and 74.8% of the polymer was in two components of mol. wt lO,OOO-50,000.The majority of the organic Pin HA-PE was associated with the medium molecular weight fractions (79.2%) while 16.8% of the P was associated with materials possessing mol. wt < 10,000. Attempts to demonstrate the presence of organic P functional groups contained in HA-PE by infrared spectroscopy was limited by the relatively small amounts of organic P incorporated into the model humic polymers. The results obtained show that a portion of the unidentified organic P in soil humic substances may arise from the inco~oration of organic compounds wntaining both amino and phosphate ester functional groups during oxidative ~lymerization of polyphenols.
INTRODUCTION
The chemical nature of soil organic P has not been fully elucidated although several forms of organic P have been identified including inositol-P esters, phospholipids and nucleic acids. Various studies indicate that <60x of soil organic P is present as known biochemicals such as inositol phosphates (Anderson, 1980). Organic P has been detected in the humic fraction of soils, especially in high molecular weight fractions (Dormaar, 1972; Moyer and Thomas, 1970; Swift and Posner, 1972; Thomas and Bowman, 1966). Typically, >40% of the total organic P in soils is associated with the fulvic and humic fractions. This association warrants investigation as a probable source for some of the uncharacterized organic P in soil. Nitrogenous compounds containing free -NH, groups have been covalently linked, via nucleophilic addition, to aromatic rings of synthetic humic polymers (Haider et al., 1965; Flaig, 1966; Ladd and Butler, 1966a, b; Haider and Martin, 1967, 1970; Martin and Haider, 1969; Bondietti et ai., 1972). Phospho~lated organic compounds containing a free -NH2 group may also be incorporated into humic substances through a mechanism similar to that suggested for the incorporation of organic N. Organic P compounds incorporated during the formation of humic molecules would be an integral part of the humic structure.
Our objective was to demonstrate that organic P compounds can be covaientiy Linked into model humic polymers prepared by oxidative polymerization of poiyphenois. The characteristics of organic P in the model humic polymers were evaluated by elemental, functional group, solubility, gel filtration and infrared spectroscopic analyses.
213
MATERIALS AND METHODS
Preparation
of model
humic polymers
Model humic polymers were synthesized in the presence and absence of low molecular weight N or P compounds by chemical oxidation of a mixture of aromatic compounds (Table 1). To insure that organic P was covalently bonded to humic polymers, contamination of polymers with P was minimized by eliminating phosphate buffers and by using 0, as the oxidant in the absence of mushroom tyrosinase, which was found to contain P. Phosphate present in either a buffer or tyrosinase could become adsorbed during the synthesis of humic polymers, resulting in overestimation of organic P contained in the model polymers. Therefore, model humic polymers were prepared by chemical rather than enzymatic oxidation. For each polymer, 3 mmol of each of the phenolic compounds listed in Table 1 were added to 2.5 1 of deionized H,O and the solution adjusted to pH 7 with 6 N NaOH to ensure dissolution of all
214
CHRISTINE A.
BRANNON and LEEE. SOMMERS
Table 1. Aromatic compounds used to synthesize model humic polymers Toluenes 2,3-Dihydroxytoluene 2,6-DihydroxytoIuene 3,4-Dihydroxytoloene 3,SDihydroxytoluene
(4-homopyr~at~hoi) (orcinol)
Cinnamic acids o-Coumaric m-Coumaric p-Coumaric 3,4-Dihydroxy~innami~ acid (caffeic) 4-Hydroxy,3-methoxycinn~ic acid (ferulic) Benzenes phenols 1,3-Dihydroxybenzene (resorcinol) 1,2,3-Ttihydroxybenzeae (pyrogallol) 1,3,5-Ttihydroxybenzene (phloroglucinol) Benzoic acids o-Hydroxybenzoic acid (salicyclic) ~-Hydroxy~nzoic acid p-Hydroxybenzoic acid 2,3-Dihydroxybenzoic acid (o-pyrccatechuic) 2,4-Dihydroxybenzoic acid (/?-resorcyclic) 2,5-Dihydroxybenzoic acid (gentistic) 2.6-Dihvdroxvbenzoic acid fy-resorcvclic) 3:CDih;drox&enzoic acid (~rotocakchtkc) 3,5-Dihydroxybenzoic acid (a-resorcyclic) 2,4,6-T~hydroxy~nzoic acid 3,4,5-T~hydroxy~nzo~c acid (gallicf 4-Hydroxy,3-methoxybenzoic acid (vanillic) 4-Hydroxy,3,5-dimethoxybenzoic acid (syringic) Amino compounds Swine (HA-S) Ethanolamine (HA-E) Phosphate compounds KWQ, Cilycerophosphate (HA-GP) Phosuhoserine (HA-P9 Phosbboethanoiamine (HA-PE) “Abbreviation
for synthetic polymer prepared.
reagents. The desired amino or phosphate compound (18 mmol) was then added to the mixture of polyphenols. The solution was adjusted to pH 8.5 with 3 N NaOH and aerated for 7 days at room temperature (22”C), by which time the solution had turned dark brown or black. The suspension of synthetic humic material was concentrated to approximately 500ml in a forced-air oven (7o”C), thoroughly dialyzed against frequent changes of deionized H,O and concentrated to a small volume at 70°C. The humic polymers were freeze-dried and stored at room temperature in the dark. The abbreviations used for each model humic polymer are shown in Table 1 and they will be used throughout this section. The control polymer was prepared by oxidizing the aromatic compounds without any N or P amendments. Characterization
qf model humic polymers
Total C content was determined by wet oxidation (Nelson and Sommers, 1974), total N by the microKjeldahl method (Nelson and Sommers, 1972) and total P by calorimetry (Murphy and Riley, 1962) after perchloric acid digestion (Sommers and Nelson, 1972). The total N and P content was also determined for the N and P compounds used to evaluate the purity of the reagents used. Total acidity was determined by the Ba(OH), method of Schnitzer and Gupta (1965). Carboxyl content was estimated by the modified calcium acetate method of Woltzclaw and
Sposito (1979). Total hydroxyl content was taken as the difference between total acidity and carboxyl content. Moisture and ash content were determined gravimet~c~ly after drying at 105°C for 15 h and heating in a mufIle furnace at 650°C for 4 h, respectively. The Ed/E6 ratios were determined by dissolving 0.5 mg of model humic polymer in 25 ml of 0.05 N NaHC03 and measuring the absorbance at 465 and 665 nm on a Beckman Model 24 spectrophotometer. Infrared spectroscopy (IR) was used to determine the presence of functional groups in model humic polymers containing organic P. Pressed KBr discs were prepared by mixing 0.5 mg of phosphoethanolamine (PE), HA-E or HA-PE with 300 mg of ground KBr in a mortar and pestle, transferring the mixture to a KBr die and applying 455 MPa of pressure under a vacuum for 15 min. The IR spectra were obtained with a Perkin-Elmer Model 180 double-beam grating spectrometer. The percent transmittance and corresponding wavenumber (4000-250 cm-‘) were recorded for each OScm-’ interval and stored on cassette tapes. Before plotting with a Houston Instruments X-Y plotter, all IR spectra were smoothed using a 25 point mathematical routine. A blank KBr disc was placed in the reference beam for all analyses. The following references were used in making infrared band assignments: Bellamy (1975); Sadtler SIPS (1967); White (1964); Alpert et al. (1970); Filip et al. (1974); Stevenson and Goh (1971). The distribution of organic P in model humic polymers was determined by physical and chemical fractionation. The model humic polymer HA-PE was fractionated by gel filtration to estimate molecular weight and P distribution. A sample of humic acid (acid-insoluble, alkali-soluble) extracted from a Chalmers silt loam soil (fine-silty, mixed, mesic, Typic Haplaquoll) was also analyzed by gel filtration. Sephadex G- 100 (Pharmacia Fine Chemicals, Uppsala, Sweden) was poured into a glass column (2.5 x 100 cm). Tris (2-amino-2(hydroxymethyl)propane- 1,3-diol) buffer (50.14 g Tris f 4.2 ml cont. HCl 1-l at pH 9) was used as the eluant, as suggested by Swift and Posner (1971) to overcome the gel-solute interactions frequently observed in gel filtration of humic materials. Humic acid samples (10 mg) were dissolved in 1 ml Tris and loaded onto the column, Effluent was collected (flow rate of 42 ml h-*) and the absorbance of each 1Oml fraction was measured at 410nm and an aliquot was analyzed for total P following perchloric acid digestion (Sommers and Nelson, 1972). The amount of P was obtained by referring to a standard curve (O-5 ,ug P) prepared from a standard KH,PO, solution. The Sephadex G-100 column was standardized with Blue Dextran 2000 (Pharmacia Fine Chemicals) and methyl organge (mol. wt 327). The amount of Blue Dextran and methyl orange contained in each fraction was determined at 610 and 510 nm, respectively using a Beckman Model 24 spectrophotometer. The mode1 humic polymer HA-PE was fractionated into the humic acid (acid insoluble, alkali soluble) and fulvic acid (acid soluble, alkali soluble) components. Freeze-dried HA-PE (approx. 0.3 g) was dissolved in 5 ml of 0.5 N NaOH, adjusted to pH 1 with cont. HCl, equilibrated for 16 h and centri-
215
Preparation of model humics containing organic P Table 2. Elemental and functional group analysis of model humic polymers” Total Compound added during humic synthesi@ None KH,PO, Glycerophosphate Serine Phosphoserine Ethanolamine Phosphoethanolamine
Apparent yield’ (g) 4.2 4.0 2.7 3.3 4.4 3.6 5.6
&I% ratio
Ash
5.36 -6.19 5.51 5.34 5.54 5.57 5.59
6.97 9.81 9.22 12.69 11.23 6.37 13.90
.~
N -~
--F%) $4.8 55.3 53.5 55.6 53.8 50.6 56.8
P
0.03 0.04 0.09 0.70 0.20 1.65 0.56
--.
0.006 0.002 0.016 0.002 0.254 0.001 0.942
Total acidity p( 10.46 9.88 10.68 8.70 7.86 8.62 11.87
Total Carboxyld hydroxyP’ m-equiv g-I)------~__ 1.56 8.90 1.60 8.22 1.73 8.95 2.00 6.79 1.47 7.86 I.60 6.89 1.77 10.00
*All values reported on a moisture and ash-free weight basis. bRefer to Table I for listing of compounds used to synthesize model humic polymers. ‘g product recovered following lyophilization. dMethod of Holtzclaw and Sposito (1979). ‘Total hydroxyl content determined by difference (total acidity--COOH).
fuged. The humic acid precipitation process was repeated and the final solid material rinsed with deionized H,O. The supernatants from all the centrifugation were pooled to yield the fulvic acid fraction, Subsamples of the HA-PE model polymer and the precipitated humic acid were then analyzed for total C, N and P. The C, N and P contents of the fulvic acid fraction were determined by difference between the original polymer and the humic acid fraction. RESULTS
Characterization of model humic polymers The chemical oxidation of polyphenolic mixtures in the presence and absence of P-containing compounds yielded between 2.7 and 5.6 g humic polymer (Table 2). The greatest quantity of humic polymer was formed in the presence of compounds containing both amino and phosphate groups, i.e. 4.4g for HA-PS and 5.6 g for HA-PE. Decreased yield of humic polymers was found for systems amended with serine and ethanolamine (3.3-3.6 g). Humic polymers prepared in the absence of organic P or N compounds (control and KH,PO,) yielded similar amounts of product (-4 g) while the least amount of model humic polymer was synthesized in the presence of glycerophosphate (2.7 g). The organic C content of the model humic polymers ranged from 50.6 (HA-E) to 56.8% C (HA-PE) and averaged 54.3% C. Model humic polymers synthesized in the presence of non-nitrogenous compounds (control, KH2P04 and HA-GP) contained no organic N. The organic N content of humic polymers prepared in the presence of non-phosphorylated N compounds was somewhat lower than expected, ranging from 0.70 (HA-S) to 1.65% N (HA-E). A comparison of the N content in polymer HA-PE vs HA-E and polymer HA-PS vs HA-S indicated that the presence of phosphorylated analogues decreased the N content of the synthetic humic polymers by 66.1 and 71.4x, respectively. Model humic polymers synthesized in the presence of KH,PO.,, glycerophosphate, serine and ethanolamine contained no organic P. The humic polymers prepared in the presence of phosphorylated N compounds contained 0.254 and 0.942% P for HA-PS and HA-PE, respectively. Total acidity of the model humic polymers ranged from 7.86 to 11.87 m-equiv g-‘. Carboxyl contents ranged from 1.47 to 2.00 m-equiv g-’ while hydroxyl contents ranged from 6.79 to 10.00 m-
equiv g-‘. The ash content of the model humic polymers was somewhat higher than expected, ranging from 6.4 (HA-E) to 13.9% (HA-PE). The preparations were not acidified prior to dialysis and thus Na + was likely associated with the functional groups. As expected, the E,/E6 ratio was similar for all model polymers ranging from 5.34 to 6.19, indicating similar molecular complexity of the polymers. The N or P content of the model polymers along with yield data enable calculation of the proportion of added N or P incorporated into the polymers. Greater proportions of ethanolamine were incorporated into model humic polymers than serine as reflected by 17.5 and 5.7% of the added N being found in HA-E and HA-S, respectively. In comparison, only 5.6 and 1.3% of the added organic P was incorporated into HA-PE and HA-PS, respectively. It should also be noted that the molar ratio of N/P in HA-PS and HA-PE is greater than the 1:1 ratio of the PS and PE added to systems for synthesis of model humics indicating that there was some hydrolysis of phosphate esters during the in~~oration of PS and PE into model humics. The overall similarity in major IR absorption bands of synthetic humic polymers with and without PE was expected since they were both synthesized from the same polyphenolic precursors (Fig, 1). A striking feature of both spectra is the strong and very broad absorbance band centered around 3400 cm-‘. This band arises from the H-bonded OH stretching mode arising from COOH, phenol&OH and polymeric intermolecular H-bonded OH groups. The infrared spectra of the model humic polymers formed through the chemical oxidation and subsequent polymerization of polysubstituted phenolic subunits resemble previously published spectra of model phenolic polymers (Bondietti et al., 1972; Filip et d., 1974; Mathur and Schnitzer, 1978; Wang et al., 1978); fungal polymers (Filip er al., 1974) and soil humic substances (Schnitzer and Gupta, 1964; Stevenson and Goh, 1971; Schnitzer and Khan, 1972; Filip et al., 1974; Tan, 1976; Eltantawy and Baverez, 1978). The aromatic C-N stretching vibration characteristic of secondary aromatic amines at 1275 cm-’ indicates the presence of N in the model humic polymers. Although secondary aromatic amine N-H stretching vibrations occur in the vicinity of the 1275 cm-’ band (Bellamy, 1975), they are generally overlaid by the stronger aromatic C-N
CHRISTINE
2750 75
1 2125
Wovenumber
1500
I .9+5
A. BRANNON and LEE E. SOMMERS
250
(cm-‘)
Fig. 1. Infrared spectra of model humic polymers. A, 0.5 mg of HA-E polymer and B, l.Omg of HA-PE polymer. absorbances. The C-O stretching vibrations and OH deformations of COOH may also account for the 1275 cm-’ band since they characteristically absorb between 1320 and 1211 cm-‘. Assignments for the major infrared absorption bands of the model humic polymers are summarized in Table 3. Organic P incorporat:d into model humic polymers as PE was examined with IR. The infrared spectra were recorded of (1) PE; (2) model humic polymer containing PE (HA-PE) and; (3) for humic polymer containing E (HA-E). A sharp well-defined spectrum was obtained for PE while diffuse, broad peaks were obtained for both model humic polymers. The absorption bands corresponding to organic P functional groups of PE were: P=O, free (1256 cm-‘); P=O, H-bonded (1168 cm-’ and P-O-C (1185, 1032, 945 and 770cm-‘). The P-OH absorbantes were broad, shallow bands between 2560 and
2700cm-’ (Bellamy, 1975) and are of little use in identifying phosphorylated compounds. Infrared absorption bands characteristic of organic P functional groups were not detectable in the spectrum of HA-PE. Gel chromatography confirmed the polydisperse nature of model humic substances. The continuous elution pattern exhibited by both the model humic polymer (HA-PE) and the soil humic acid indicated that molecular weights ranged from ~2500 to > 100,000. Gel filtration of HA-PE indicated the presence of three major mol. wt species: a component with mol. wt > 100,000 and two medium mol. wt components (15,000 and 20,000 (Fig. 2)). Based on absorbance at 410nm, most of the HA-PE humic polymer was distributed in components II and III. Comparatively little of the HA-PE polymer was found in fraction I. The distribution of molecular weight components differed for synthetic and soil humic substances (Table 4). The soil humic acid contained greater amounts (16.0%) of high mol. wt (> 50,000) constituents compared with the model humic polymer (2.3%). The model humic polymer was composed predominantly of medium mol. wt (lO,OOO-50,000) components (74.8%) as compared to the soil humic acid (37.8%). The soil humic acid contained a larger proportion of low mol. wt (< 10,000) constituents (46.7%) than the model humic polymer (22.9%). The distribution of P for HA-PE and soil HA also differed considerably (Fig. 2). Most of the P contained in HA-PE (79.2%) was associated with the medium mol. wt (1O,OOO-50,000)fractions while very little P (3.9%) was detected in the high mol. wt (> 50,000) fractions (Table 4). In contrast, the distribution of P contained in the soil HA was shifted towards the higher mol. wt (>50,000) fractions (39.7%) with a considerable amount of P (15.90/,) associated with the > 100,000 mol. wt fraction. Interestingly, although a sizeable proportion of the soil HA (46.7%) was found in low mol. wt (-c 10,000) fractions, only trace amounts of the P were associated with these fractions. In contrast, a similar proportion of the total polymer and total P was associated with low mol. wt fractions (22.9 and 16.83/ respectively)
Table 3. The main IR absorption bands of the model humic polymers HA-E and HA-PE Frequency (cm-‘) .__ 3400 2925
Assignment
Intensity’
Polymeric OH stretch, H-bonded vs, broad C-H stretch of benzene and w to m phenolic-CH, 2925, 2860 Aliphatic C-H stretch of CH, w to m 1700 C=O stretch of aryl acids m 1612 Aromatic skeletal ring breathing YS mode; C=C conjugated to C=O; COO1515 Aromatic skeletal ring breathing mode m 1455 Aromatic skeletal ring breathing mode; sh aliphatic C-H deformation of CH, 1387 C-O stretch and OH deformation of S phenolic-OH; COO1275 C-N stretch of secondary aromatic amine; m N-H stretch of secondary aromatic amine; C-O stretch and OH deformation of COOH 1167 C-O stretch and OH deformation of COOH w 1080 Aromatic C-O stretch and OH deformations w 1030 (0-CH,?) w 800 Aromatic C-H out-of-plane deformations m ‘vs, very strong; s, strong; m, medium; w, weak; sh, shoulder.
217
Preparation of model humics containing organic P 0.5 c
r
10
25,000
Void volume
15
20
25
30
35
10.000
40
45
Fraction
50
55
60
65
70
75
00
number
Fig. 2. Fractionation of HA-PE (-) and humic acid isolated from Chalmers soil (---) on Sephadex G-100. Humics dissolved in 1 ml of 0.41 M Tris were applied to 2.5 x 100cm column of Sephadex G-100 and eluted with 0.41 M Tris. Ten ml fractions were collected at a flow rate of 42 ml h-’ and analyzed for P and absorbance at 410 nm. of HA-PE. About 99% of the P applied to the Sephadex G-100 column was recovered indicating that HA-PE was not adsorbed onto the gel matrix in the presence of the Tris buffer eluant. The P content of the soil HA was estimated to be 0.87% P which favorably compares with the 0.73% P content of HA-PE. The model humic polymer HA-PE was separated into humic and fulvic acid fractions on the basis of solubility in acid. The majority of the C (83.2x), N (79.8%) and P (75.3%) originally contained in HA-PE was found in the acid insoluble humic acid fraction. The molar N/P ratios of HA-PE and the resulting humic acid indicated that the C-O-P bonds in HA-PE were stable to chemical hydrolysis during the humic-fulvic acid fractionation procedure.
DISCUSSION
Infrared spectroscopy was not particularly informative in the detection of organic P functional groups in the model humic polymers. Either the concentrations of organic P were below the detection limits of the infrared techniques employed or the absorbantes normally assignable to organic P were obscured by stronger or broad absorptions attributed to other functional groups in the model humic polymers. Two major elution bands corresponding to high mol. wt and low mol. wt components were found
for model humic substances. The elution pattern observed in the gel filtration of the soil humic acid agrees with the gel chromatography studies of soil humic substances of Moyer and Thomas (1970), Swift and Posner (1971) and Tan (1976). The bimodal elution pattern observed from the gel filtration of the HA-PE model humic polymer yielded mol. wt fractions which differed from the anticipated mol. wt distribution characteristic of soil humic substances. The dominance of medium mol. wt (10,00~50,000) constituents and the relative absence of high mol. wt (> 50,000) components could be expected during the synthesis of HA-PE because simple polyphenols underwent chemical oxidation and subsequent polymerization into humic substances. Within one week, the phenols had been transformed into low (< 10,000) and medium (lO,OOO-50,000) mol. wt polycondensates. Only a small amount of high mol. wt (> 50,000) material would be expected to be formed within this period, especially in the absence of polymerizing enzymes (e.g. phenolase) coupled with the non-availability of larger organic compounds (e.g. proteins, carbohydrates) commonly found in plant or animal residues. The relative absence of high mol. wt constituents in synthetic humic polymers compared with soil humic acids was reported in the gel filtration studies of Ladd and Butler (1966b) and Haider and Martin (1970). Haider and Martin (1970) demonstrated that high mol. wt fractions of synthetic phenolic humic polymers could be increased up to 6%
Table 4. The molecular weight and phosphorus distribution in synthetic humic containing phosphoethanolamine (HA-PE) and soil humic acid as determined by fractionation on Seohadex G-100 Percent of weight Molecular weight fraction > 100,000 50,00&100,000 25,OOC-50,000 10,00&25,000 < 10,000
HA-PE 0.5 1.8 22.1 52.1 22.9
Soil humic acid 1.3
a.1 9.5 27.8 46.1
Percent of phosphorus HA-PE
Soil humic acid
1.1 2.8 21.4 57.8 16.8
15.9 23.8 38.5 21.8 trace
218
CHRISTINEA. BRANNONand LEE E. SOMMERS
through the incorporation of nitrogenous compounds such as glycine, peptone or albumin into the polymers. Model humic polymers synthesized by fungi grown on a variety of media exhibited a mol. wt distribution similar to soil humic acids (Ladd and Butler, 1966b; Martin et al., 1979; Martin and Haider, 1969; Haider and Martin, 1970). Organic P has frequently been reported in association with soil humic substances, in particular with high mol. wt fractions. The majority of organic P contained in HA-PE polymer (75.3%) was found in the humic acid fraction. Fractionation by gel filtration of the soil humic acid indicated that approximately 40% of the P was associated with high mol. wt (>50,000) components. Moyer and Thomas (1970) reported a similar distribution of organic P with 36% contained in high mol. wt (> 50,000) fractions while Thomas and Bowman (1966) observed somewhat higher proportions of organic P (74.5%) in the high mol. wt (> 50,000) fractions of humic acid. Swift and Posner (1972) also found that the majority of P in humic acids extracted from Australian soils was in association with high mol. wt fractions. About SOT/, of the P contained in the model HA-PE humic polymer was associated with medium (lO,OOO-50,000) mol. wt fractions. The distribution of Pin the various mol. wt fractions of humic substances would be expected to shift towards higher mol. wt fractions with time. Similar trends in the mol. wt distribution of both N and P in humic acids were reported by Swift and Posner (1972), supporting the mechanism advanced earlier that organic P may be incorporated into humic substances through the interaction of the free amino groups of a phosphorylated nitrogenous compound with phenolic subunits. Thus, during polymerization, organic P is first associated with low and medium mol. wt humic components. With time, the organic P becomes associated with higher mol. wt fragments as the phenolic constituents continue to condense and incorporate other available organic compounds. Easily-degradable organic P would be microbially decomposed during the early stages of humus formation. The resulting highly-resistant organic P fraction associated with high mol. wt components of humic acid would remain essentially non-available to both plants and microorganism. In summary, this study demonstrated that phosphate esters can be incorporated into humic materials if the organic P compound contains a free amino group. Martin, Haider and others (Haider et al., 1965; Flaig, 1966; Ladd and Butler, 1966a, b; Haider and Martin, 1967, 1970; Martin and Haider, 1969; Bondietti et al., 1972) have shown that compounds containing -NH, functional groups undergo nucleophilic addition to activated phenolic compounds. Thus, one mechanism by which organic P may become associated with the soil humic fraction involves incorporation of phosphorylated organic compounds group during oxidative containing a free -NH, polymerization of polyphenols. Organic P compounds thus incorporated would become an integral part of the soil humic material and this mechanism may explain the character of a portion of the currently undefined organic P associated with soil orgamc matter.
REFERENCES Alpert N. L., Keiser W. E. and Szymanski H. A. (1970) Theory and Practice of Infrared Spectroscopy.
Plenum
Press, New York. Anderson G. (1980) Assessing organic phosphorus in soils. In The Role of Phosphorus in- Agri<u~e (F. E. Kha-
sawneh. E. C. SamDle and E. J. Kammath. Eds).
PP.
411-43i. American’ Society of Agronomy, Ma&s&, Wisconsin. Bellamy L. J. (1975) The Infrared Spectra of Complex Molecules. Wiley, New York. Bondietti E., Martin J. P. and Haider K. (1972) Stabilization of amino sugar units in humic type polymers. Soil Science Society of America Proceedings 36, 591-602.
Dormaar J. F. (1972) Chemical properties of organic matter extracts from a number of A, horizons by a number of methods. Canadian Journal of Soil sciences 52, 67-71. Filip Z., Haider K., Beutelspacher H. and Martin J. P. (1974) Comparisons of IR-spectra from melanins of microscopic soil fungi, humic acids and model phenol polymers. Geoderma 11, 31-52. Flalg W. (1966) The chemistry of humic substances. In The Use of Isotopes in Soil Organic Matter Studies, pp. 103-127. Pergamon Press, Oxford. Haider K. and Martin J. P. (1967) Synthesis and transformation of phenolic compounds by Epicoccum nigrum in relation to humic acid formation. Soil Science Society of America Proceedings 31, 766-712.
Haider K. and Martin J. P. (1970) Humic acid-type phenolic polymers from Aspergillus sydowi culture medium, Stathybofrys spp. cells and autoxidized phenol mixtures. Soil Biology & Biochemistry 2, 145-156.
Haider K., Frederick L. R. and Flaig W. (1965) Reactions between amino acid compounds and phenols during oxidation. Plant and Soil 22, 49-64. Holtzclaw K. M. and Sposito G. (1979) Analvtical properties of the soluble, metal-complkxing’ fractions in sludgesoil mixtures: IV. Determination of carboxyl groups in fulvic acid. Soil Science Society of America Journal 43, 3 18-323.
Ladd J. N. and Butler J. H. A. (1966a) Comparison of properties of synthetic and natural humic acids. In The Use of Isotopes in Soil Organic Maiter Studies, pp. 143-159. Pergamon Press, Oxford. Ladd J. N. and Butler J. H. A. (1966b) Comparison of some properties of soil humic acids and synthetic phenolic polymers incorporating amino derivatives. Australian Journal of Soil Research 4, 41-54.
Martin J. P. and Haider K. (1969) Phenolic polymers of Stachybotrys atra, Stachybotrys chartarum and Epieoccum nigrum in relation to humic acid formation. Soil Science 107, 260-270.
Martin J. P., Haider K. and Linhares L. F. (1979) Decomposition and stabilization of ring “‘C-labeled catechol in soil. Soil Science Society of American Journal43, 10&104. Mathur S. P. and Schnitzer M. (1978) A chemical and spectroscopic chracterization of some synthetic analogues of humic acids. Soil Science Societv_ of” America Journal 42, 591-596.
Moyer J. R. and Thomas R. L. (1970) Organic phosphorus and inositol phosphates in molecular size fractions of a soil organic matter extract. Soil Science Society of America Proceedings 34, 80-83.
Murphy J. and Riley J. P. (1962) A modified single solution method for the determination of phosphate in natural waters. Analytica Chimica Acta 27, 31-36. Nelson D. W. and Sommers L. E. (1972) A simple procedure for estimation of total nitrogen in soils and sediments. Journal of Environmental Quality 1, 423425.
Nelson D. W. and Sommers L. E. (1974) A rapid and accurate procedure for estimation of organic carbon in soils. Proceedings of Indiana Academy of Sciences 84, 456-462.
Preparation of model humics containing organic P Sadtler Standard Infrared Prism Spectra (1976) Sadtler Research Laboratories, Philadelphia. Schnitzer M. and Gupta U. C. (1964) Some chemical characteristics of the organic matter extracted from the 0 and B2 horizons of a gray wooded soil. Soil Science Society
of America
Proceedings
28, 374-377.
Schnitzer M. and Gupta U. C. (1965) Determination acidity in soil organic matter. Soil Science Society America
Proceedings
of
29, 272-277.
36, 902.-904.
Stevenson F. J. and Goh K. M. (1971) Infrared spectra of humic acids and related substances. Geochemica et Cosmochimica
Acta 35, 47 l-483.
Swift R. S. and Posner A. M. (1971) Gel chromatography of humic acid. Journal of Soil Science 22, 237-249. Swift R. S. and Posner A. M. (1972) Nitrogen, phosphorus and sulphur contents of humic acids fractionated with respect to molecular weight. Journal of Soil Science 23, SO-57.
of
Schnitzer M. and Khan S. U. (1972) Humic Substances in the Environment. Dekker, New York. Sommers L. E. and Nelson D. W. (1972) Determination of total phosphorus in soils: A rapid perchloric acid digestion procedure. Soil Science Society of America Proceedings
219
Tan K. H. (1976) Complex formation between humic acid and clays as revealed by gel filtration and infrared spectroscopy. Soil Biology & Biochemistry 8, 235-239. Thomas R. L. and Bowman B. T. (1966) The occurrence of high molecular weight organic phosphorus compounds in soil. Soil Science Society of America Proceedings 30, 799-80 1.
Wang T. S. C., Li S. W. and Huang P. M. (1978) Catalytic polymerization of phenolic compounds by a latosol. Soil Science 126, 81-87. White R. G. (1964) Handbook of Industrial Infrared Analysis. Plenum Press, New York.