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Permanganate oxidation of humic acids extracted from a gray wooded soil under different cropping systems and fertilizer treatments
Geoderma Elsevier Publishing Company, Amsterdam - Printed in The Netherlands
PERMANGANATE OXIDATION OF HUMIC ACIDS EXTRACTED FROM A GRAY WOODED SOIL UNDER DIFFERENT CROPPING SYSTEMS AND FERTILIZER TREATMENTS 1 S.U. KHAN and M. SCHNITZER
Soil Research Institute, Canada Department of Agriculture, Ottawa, Ont. (Canada) (Received April 20, 1971) ABSTRACT Khan, S.U. and Schnitzel M., 1972. Permanganate oxidation of humic acids extracted from a gray wooded soil under different cropping systems and fertilizer treatments. Geoderma, 7:113-120. Long-term effects of two cropping systems and of manure, fertilizer and lime treatments on products resulting from the permanganate oxidation of humic acids extracted from a gray wooded soil were investigated. The major oxidation products were benzenecarboxylic and phenolic acids. Humic acids extracted from soils under a 5-year rotation of grains and legumes yielded, per unit weight, more benzenecarboxylic acids than did those originating from soils under a wheat-fallow sequence. By contrast, the type of cropping system did not seem to affect the yields of phenolic acids. The application of lime and manure tended to reduce the yields of benzenecarboxylic and phenolic acids, suggesting that these treatments degraded the "cores" or more resistant chemical structures of humic acids. Other useful indexes of the relative molecular complexities of the humic acids were the total yields of oxidation products. These, regardless of rotation, were highest for the check plots and lowest for the plots treated with lime. Between 75 and 90% of the oxidation products were identified. The data show that the type of rotation, and especially the application of lime and manure, have significant effects on the synthesis and degradation of humic acid "nuclei", These effects may be brought about by increased microbial activity and/or by chemical means. Permanganate oxidation may thus serve as a guide for assessing the degree of humification of soil humic acids.
INTRODUCTION The long-term effects o f cropping systems, lime, manure, and fertilizers on the physical and chemical properties o f a gray w o o d e d soil f r o m Alberta have been under investigation for a n u m b e r o f years ( T o o g o o d and Lynch, 1959; Khan, 1969, 1970). In a previous paper (Khan, 1970) a n u m b e r o f chemical characteristics o f h u m i c acids ( H A ) e x t r a c t e d from this soil were described. Analytical m e t h o d s for the separation and identification o f products resulting f r o m the oxidative degradation o f h u m i c substances, and involving gas c h r o m a t o g r a p h y , mass s p e c t r o m e t r y and microinfrared s p e c t r o p h o t o m e t r y , have recently b e e n developed in this laboratory (Ogner and Schnitzer, 1971 ; Khan and Schnitzer, 1971). Thus, we were interested in applying these techniques to evaluate the effects o f manageJoint contribution of the Research Station Lacombe, Alberta, and of the Soil Research Institute (No.374).
114
S.U. KHAN AND M. SCHNITZER
ment practices on the "cores" or "nuclei" of the HA fractions, as characterized by the permanganate oxidation of methylated HA's. This degradative technique has been used for the elucidation of the main structural components of complex organic materials such as coal (Randall et al., 1938). wood (Pearl, 1967), lignin (Freudenberg et al., 1962; Freudenberg and Neish, 1968) and humic substances (Schnitzer and Desjardins, 1970). Following oxidation of each HA, the products were extracted into ethyl acetate and then separated by preparative gas chromatography into relatively pure components which were identified by matching their mass and IR spectra and gas chromatographic retention times with those of authentic specimens. MATERIALS AND METHODS Soil samples, cropping sequence, and treatments, were described previously (Khan, 1970). The soil, a Breton silt loam from the Breton plots, contained 10 to 15% clay and < 3% organic matter. These plots were established in 1930 by the University of Alberta and their history has been reported elsewhere (Toogood et al., 1962). Surface Ap horizons from plots 1,2, 3 and 6 in a 5-year rotation of grains and legumes (wheat - oats - barley - legume legume) and a wheat-fallow sequence were collected in May 1969 in the manner described previously (Khan, 1969). The fertilizer treatments are listed in Table I. The method of extraction and purification of HA's was identical to that reported earlier (Khan, 1970). TABLE I Treatments on Breton plots Plot no.
Fertilizer treatments* nil ¢4.8 metric tons manure per ha every five years Annual application to supply 11.2 kg N, 5.9 kg P, 16.7 kg K and 9.0 kg S per ha 4.5 metric tons lime per ha in 1930-32, 2.2 metric tons in 1938, 1.1 metric tons in 1948 and 1.1 metric tons in 1965
*These treatments are as of 1965 and are similax to, though not identical with, the treatments which have been used since the plots were established (Toogood et al., 1962). The methylation procedure was as follows: 1 g of HA was suspended in 10 ml of CH3 OH and methylated with an excess of diazomethane in ether generated from Diazald. The following oxidation procedure was adopted: 1 g of methylated HA was refluxed for 8 h with 250 ml of 4% (W/W) aqueous KMnO4 solution. Following oxidation, the excess KMnO4 was destroyed by careful addition of small volumes of CH 3 OH. The insoluble MnO2 was removed by filtration and washed with small aliquots of hot water. The
PERMANGANATE OXIDATION OF HUMIC ACIDS
115
filtrate plus washings was acidified to pH 2 with 6N H2 SO4 solution, transferred to a liquid-liquid extractor and extracted with 500 ml of ethyl acetate for 72 h. The extract (= oxidation product) was first dried over anhydrous Na2 SO4 and then in a rotary evaporator. To make the oxidation products sufficiently volatile for subsequent gas chromatographic and mass spectrometric analyses, it was necessary to methylate them. Thus, each oxidation product was dissolved in a small volume of methanol and methylated with an ether solution of diazomethane. Following removal of the excess diazomethane, the methylated preparations were dried and weighed. Each methylated oxidation product was separated by preparative gas chromatography (Hewlett-Packard model 402, flame-ionization detector, 1800 x 4 mm glass column packed with 5% SE-30 on Chromosorb W HMDS, 60 to 80 mesh, programmed from 150 ° to 300°C at a rate of 7.5°C/min). Materials representing the major peaks (see Fig.l) were eluted from
I0
Z
15
13
16
17
Jb
TIME (MIN)
~;
2'o
Fig. 1. Gas chromatographic separation of products resulting from the permanganate oxidation of methylated HA (check plot, 5-year rotation of grains and legumes). Numerals refer to components identified in Table III.
the gas chromatographic column, collected in capillary tubes and analyzed by mass spectrometry on a CEC model 2 1 - 4 9 0 mass spectrometer, using a heated direct inlet probe, and by IR as micro-KBr-pellets on a Beckman IR 12 spectrophotometer fitted with a beam condenser. As an additional check, each unknown was co-chromatographed (on the gas chromatograph) with the corresponding known compound. Quantitative estimates of each major component were made by triangulation. All solvents were purified by distillation through high-efficiency columns. Blanks were run for each solvent and, when required, corrections were made for impurities in the solvents.
116
S.U. KHAN AND M. SCHNITZER
The origins and preparations of the known compounds used as standards for the mass spectrometric and IR spectrophotometric identification of the unknowns were identical to those described earlier (Ogner and Schnitzer, 1971). C and H were determined by dry combustion and OCH3 by the Zeisel method. RESULTS AND DISCUSSION
Effect of methylation on C, H and OCH3 contents of HA's In both rotations manure gave HA's with the highest QCH3-content (Table II). Lime tended to increase the C-content of the unmethylated HA's, especially in the grains-legumes rotation. The methylated HA's contained more C and H than did the unmethylated ones. Methylation increased the OCH 3 content from 1.52 to 2.28% to between 16.65 and 17.65%. TABLE I1 Elementary composition and methoxyl content of HA's (%; dry-ash free basis) Unmethylated HA C
H
Methylated HA OCH s
C
H
OCH s
Five-year rotation of grains and legumes: Check
53.4
4.9
1.5
58.0
5.9
17.5
Manure
53.7
5.0
2.3
57.5
5.9
17.6
NPKS
54.9
5.1
1.6
57.7
6.3
17.1
Lime
57.4
4.8
1.8
59.9
6.6
17.0
Wheat-fallow sequence: Check
54.4
4.6
1.8
58.8
6.8
16.7
Manure
55.4
4.0
2.2
59.7
6.0
17.5
NPKS
55.7
4.5
1.7
59.3
6.2
17.6
Lime
56.1
4.4
1.6
59.6
6.9
16.7
Separation and identification of oxMation products The gas chromatographic separation of the products resulting from the oxidation of methylated HA extracted from the check sample of the grains and legumes rotation is illustrated in Fig. 1. Gas chromatographic separations o f the HA's extracted from the other soil samples were qualitatively similar to that shown in Fig.1. The results are summarized in Table III. The upper part o f the table lists fully methylated benzenecarboxylic acids,
PERMANGANATE OXIDATION OF HUMIC ACIDS
117
e~
Z
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•~
Z
o o: o
¢-q
< =o
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"o
"o
~
~
4
d
d
g
d
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d
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~
~
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6 Z
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118
S.U. KHAN AND M. SCHNITZER
the lower part, fully methylated phenolic acids. In addition to the compounds shown, we also isolated and identified small amounts of dimethyl esters of glutaric, adipic, pimelic, 1,2,3-propanetricarboxylic, suberic, azelaic and 1,2,3,4-butane-tetracarboxylic acids. In view of the relatively low OCH3-content of the unmethylated HA's (Table II), it is likely that most COOH and phenolic OH groups were methylated in the laboratory, so that it may be more realistic to refer to the oxidation products as benzenecarboxylic and phenolic acids rather than as esters and ethers.
Effects o f cultural and fertilizer treatments on compounds isolated The permanganate oxidation of all HA's yielded two types of major products (Table llI): (a) benzenecarboxylic acids and (b) phenolic acids. Chemical structures of the compounds that we isolated and were able to identify are listed in Fig.2.
1.
al--R2=C02CH 9 , R3=a4=RS=Wo=H
2. al=a3=co2cH 3 , R2=a~=~=ae--a
R6 i~
a2 a3
9.
Rl=a~=coaca3 , R2=rt3=as=a6=a
4. 5.
rk--a2=a3~02ca3 , a4--as=a6=H rtl=~--r~z=co2cR3 , %=aS--a6=a
6. al=rt3=aS--coaea3 , a2=rtZ=a6~ 7.
RI=R3=R5=CO2CH3 , R2=O~H3 , R4=R6=H
s.
al=~=a3=coacH3 , aS=oea3 , a4=a6=a
J.o.
~l=a2--a3=a4=co2ca3 , aS--a6=a
1.!.. ~=az=R4=rts=coacn3 , •=a6=H 12. al=R2=R3=a5---C02CH 3 , R4=%=H 13. al=rtz=a3=a4=cO2CH3 , ~=0CH3 , a6=H 1,~. 16.
RI=R2=R3=R4=R5=C02CH3 , R6=H RI=R2=R3=R4=R5:=CO2Cti3 , R6=0CH3
17.
r~l=~--a3 =R4=aS=ae=CO2CH9
co2c~3
c02c~3
i8.
ecH3
oc~3
Fig.2. Chemical structures of compounds identified.
The HA's extracted from the soils subjected to the 5-year rotation of grains and legumes produced, per unit weight, more benzenecarboxylic acids than did those originating from the wheat-fallow sequence (Table III). In the former, benzenecarboxylic acids accounted
PERMANGANATE OXIDATIONOF HUMICACIDS
119
for between about 65 and 69% of the total yield of oxidation products, while in the latter these acids constituted between approximately 55 and 60% of the total yield. By contrast, the type of cropping system did not seem to affect the yields of phenolic acids, which averaged about 20% of the total products (Table III). The most abundant benzenecarboxylic acids isolated from HA's resulting from both cropping systems were 15, 10 and 5. The grains-legumes rotation was more favourable for the synthesis of structures yielding these compounds on oxidation than was the wheat-fallow sequence. A comparison of the data for the check plots with those receiving different fertilizer treatments indicates that the latter reduced the chemical complexity of the HA's if we accept, as has been reported (Randall et al., 1938), that benzenecarboxylic acids usually arise from the oxidation of more complex chemical structures. The most effective treatments for this purpose seemed to be the application of lime. Fertilizer treatments were less effective in lowering the yields of phenolic than of benzenecarboxylic acids. Again, lime was most efficient in lowering the proportion of HA structures yielding phenolic acids on oxidation. In the case of the wheat-fallow sequence, the application of manure had a similar but less pronounced effect. Other useful indexes of the relative molecular complexities of the HA's extracted from soils subjected to the different treatmentswere the total yields of oxidation products. These, regardless of cropping system, were highest for the check plots, considerably lower for the manure treatments and lowest where lime was applied. Especially interesting is the isolation of compound 18, which was the only biphenyl compound that we were able to identify and which most likely originated from lignin (Freudenberg et al., 1962). It is especially noteworthy that by means of the analytical techniques that we have employed we were able to identify between approximately 75 and 90% of the oxidation products. We believe that the general approach described in this paper adds a new dimension to soil chemistry by directly relating the effects of cropping systems and fertilizer treatments to the "cores", that is, the more resistant chemical structures of HA's. The data presented herein show that the type of rotation, and especially the application of lime and manure, have significant effects on the synthesis and degradation of these structures. Whether the resulting increased degradation is brought about by enhanced microbiological activity, due to these treatments, and/or by chemical means awaits further investigation. The data reported are in general agreement with those obtained previously (Khan, 1970) which showed that the wheat-fallow sequence as well as lime, and to some extent manure applications, increased the neutralization capacity, especially the COOH group content, of the same HA's. These characteristics may be taken as indexes of increased humification. Thus, a low yield of products resulting from the permanganate oxidation of HA's may be indicative of a relatively high degree of humification, so that permanganate oxidation may serve as a guide for assessing the degree of humification of soil HA's.
120
S.U. KHAN AND M. SCHNITZER
ACKNOWLEDGEMENTS We are i n d e b t e d to the D e p a r t m e n t o f Soil Science, University o f Alberta, for allowing us to use soils f r o m the Breton plots. We are grateful to D. Friessen and J.G. Desjardins for technical assistance. REFERENCES Freudenberg, K., Chen-Loung, C. and Cardinale, G., 1962. Die Oxydation des methylierten and naturlichen Lignins. Chem. Ber., 95: 2814-2828. Freudenberg, K. and Neish, A.C., 1968. Constitution and Biosynthesis of Lignin. Springer Verlag, New York, N.Y., 79 pp. Khan, S.U., 1969. Some carbohydrate fractions of a gray wooded soil as influenced by cropping systems and fertilizers. Can. J. Soil. SCL, 49: 219-224. Khan, S.U., 1970. Enzymatic activity in a gray wooded soil as influenced by cropping systems and fertilizers. SoilBiol. Biochem., 2: 137-139. Khan, S.U., 1970. Humic acid fraction of a Gray Wooded Soil as influenced by cropping systems and fertilizers. Geoderma, 3: 247-254. Khan, S.U. and Schnitzel M., 1971. Further investigations on the chemistry of fulvic acid, a soil humic fraction. Can. J. Chem., 49: 2302-2309. OgneL G. and Schnitzer, M., 1971. The chemistry of fulvic acid, a soil humic fraction, and its relation to lignin. Can. J. Chem., 49: 1053-1063. Pearl, I.A., 1967. The Chemistry ofLignin. Dekker Inc., New York, N.Y., 199 pp. Randall, R.B., Benger, M. and Groocock, C.M., 1938. The alkaline permanganate oxidation of organic substances selected for their bearing upon the chemical constitution of coal. Proc. Roy Soc. Lond., Ser. A, 165: 432-452. Schnitzer, M. and Desjardins, J.G., 1970. Alkaline permanganate oxidation of methylated and unmethylated fulvic acid. SoilSci. Soc. Am. Proc., 34: 77-79. Toogood, J.A., and Lynch, D.L., 1959. Effect of cropping systems and fertilizers on mean weightdiameter of aggregates of Breton plot soils. Can. J. Soil ScL, 39: 151-156. Toogood, J.A., Bentley, C.F., Webster, G.R. and Moore, A.W., 1962. Gray Wooded Soils and Their Management. Univ. of Alberta, Edmonton, Alta., 21 pp.
PERMANGANATE OXIDATION OF HUMIC ACIDS EXTRACTED FROM A GRAY WOODED SOIL UNDER DIFFERENT CROPPING SYSTEMS AND FERTILIZER TREATMENTS 1 S.U. KHAN and M. SCHNITZER
Soil Research Institute, Canada Department of Agriculture, Ottawa, Ont. (Canada) (Received April 20, 1971) ABSTRACT Khan, S.U. and Schnitzel M., 1972. Permanganate oxidation of humic acids extracted from a gray wooded soil under different cropping systems and fertilizer treatments. Geoderma, 7:113-120. Long-term effects of two cropping systems and of manure, fertilizer and lime treatments on products resulting from the permanganate oxidation of humic acids extracted from a gray wooded soil were investigated. The major oxidation products were benzenecarboxylic and phenolic acids. Humic acids extracted from soils under a 5-year rotation of grains and legumes yielded, per unit weight, more benzenecarboxylic acids than did those originating from soils under a wheat-fallow sequence. By contrast, the type of cropping system did not seem to affect the yields of phenolic acids. The application of lime and manure tended to reduce the yields of benzenecarboxylic and phenolic acids, suggesting that these treatments degraded the "cores" or more resistant chemical structures of humic acids. Other useful indexes of the relative molecular complexities of the humic acids were the total yields of oxidation products. These, regardless of rotation, were highest for the check plots and lowest for the plots treated with lime. Between 75 and 90% of the oxidation products were identified. The data show that the type of rotation, and especially the application of lime and manure, have significant effects on the synthesis and degradation of humic acid "nuclei", These effects may be brought about by increased microbial activity and/or by chemical means. Permanganate oxidation may thus serve as a guide for assessing the degree of humification of soil humic acids.
INTRODUCTION The long-term effects o f cropping systems, lime, manure, and fertilizers on the physical and chemical properties o f a gray w o o d e d soil f r o m Alberta have been under investigation for a n u m b e r o f years ( T o o g o o d and Lynch, 1959; Khan, 1969, 1970). In a previous paper (Khan, 1970) a n u m b e r o f chemical characteristics o f h u m i c acids ( H A ) e x t r a c t e d from this soil were described. Analytical m e t h o d s for the separation and identification o f products resulting f r o m the oxidative degradation o f h u m i c substances, and involving gas c h r o m a t o g r a p h y , mass s p e c t r o m e t r y and microinfrared s p e c t r o p h o t o m e t r y , have recently b e e n developed in this laboratory (Ogner and Schnitzer, 1971 ; Khan and Schnitzer, 1971). Thus, we were interested in applying these techniques to evaluate the effects o f manageJoint contribution of the Research Station Lacombe, Alberta, and of the Soil Research Institute (No.374).
114
S.U. KHAN AND M. SCHNITZER
ment practices on the "cores" or "nuclei" of the HA fractions, as characterized by the permanganate oxidation of methylated HA's. This degradative technique has been used for the elucidation of the main structural components of complex organic materials such as coal (Randall et al., 1938). wood (Pearl, 1967), lignin (Freudenberg et al., 1962; Freudenberg and Neish, 1968) and humic substances (Schnitzer and Desjardins, 1970). Following oxidation of each HA, the products were extracted into ethyl acetate and then separated by preparative gas chromatography into relatively pure components which were identified by matching their mass and IR spectra and gas chromatographic retention times with those of authentic specimens. MATERIALS AND METHODS Soil samples, cropping sequence, and treatments, were described previously (Khan, 1970). The soil, a Breton silt loam from the Breton plots, contained 10 to 15% clay and < 3% organic matter. These plots were established in 1930 by the University of Alberta and their history has been reported elsewhere (Toogood et al., 1962). Surface Ap horizons from plots 1,2, 3 and 6 in a 5-year rotation of grains and legumes (wheat - oats - barley - legume legume) and a wheat-fallow sequence were collected in May 1969 in the manner described previously (Khan, 1969). The fertilizer treatments are listed in Table I. The method of extraction and purification of HA's was identical to that reported earlier (Khan, 1970). TABLE I Treatments on Breton plots Plot no.
Fertilizer treatments* nil ¢4.8 metric tons manure per ha every five years Annual application to supply 11.2 kg N, 5.9 kg P, 16.7 kg K and 9.0 kg S per ha 4.5 metric tons lime per ha in 1930-32, 2.2 metric tons in 1938, 1.1 metric tons in 1948 and 1.1 metric tons in 1965
*These treatments are as of 1965 and are similax to, though not identical with, the treatments which have been used since the plots were established (Toogood et al., 1962). The methylation procedure was as follows: 1 g of HA was suspended in 10 ml of CH3 OH and methylated with an excess of diazomethane in ether generated from Diazald. The following oxidation procedure was adopted: 1 g of methylated HA was refluxed for 8 h with 250 ml of 4% (W/W) aqueous KMnO4 solution. Following oxidation, the excess KMnO4 was destroyed by careful addition of small volumes of CH 3 OH. The insoluble MnO2 was removed by filtration and washed with small aliquots of hot water. The
PERMANGANATE OXIDATION OF HUMIC ACIDS
115
filtrate plus washings was acidified to pH 2 with 6N H2 SO4 solution, transferred to a liquid-liquid extractor and extracted with 500 ml of ethyl acetate for 72 h. The extract (= oxidation product) was first dried over anhydrous Na2 SO4 and then in a rotary evaporator. To make the oxidation products sufficiently volatile for subsequent gas chromatographic and mass spectrometric analyses, it was necessary to methylate them. Thus, each oxidation product was dissolved in a small volume of methanol and methylated with an ether solution of diazomethane. Following removal of the excess diazomethane, the methylated preparations were dried and weighed. Each methylated oxidation product was separated by preparative gas chromatography (Hewlett-Packard model 402, flame-ionization detector, 1800 x 4 mm glass column packed with 5% SE-30 on Chromosorb W HMDS, 60 to 80 mesh, programmed from 150 ° to 300°C at a rate of 7.5°C/min). Materials representing the major peaks (see Fig.l) were eluted from
I0
Z
15
13
16
17
Jb
TIME (MIN)
~;
2'o
Fig. 1. Gas chromatographic separation of products resulting from the permanganate oxidation of methylated HA (check plot, 5-year rotation of grains and legumes). Numerals refer to components identified in Table III.
the gas chromatographic column, collected in capillary tubes and analyzed by mass spectrometry on a CEC model 2 1 - 4 9 0 mass spectrometer, using a heated direct inlet probe, and by IR as micro-KBr-pellets on a Beckman IR 12 spectrophotometer fitted with a beam condenser. As an additional check, each unknown was co-chromatographed (on the gas chromatograph) with the corresponding known compound. Quantitative estimates of each major component were made by triangulation. All solvents were purified by distillation through high-efficiency columns. Blanks were run for each solvent and, when required, corrections were made for impurities in the solvents.
116
S.U. KHAN AND M. SCHNITZER
The origins and preparations of the known compounds used as standards for the mass spectrometric and IR spectrophotometric identification of the unknowns were identical to those described earlier (Ogner and Schnitzer, 1971). C and H were determined by dry combustion and OCH3 by the Zeisel method. RESULTS AND DISCUSSION
Effect of methylation on C, H and OCH3 contents of HA's In both rotations manure gave HA's with the highest QCH3-content (Table II). Lime tended to increase the C-content of the unmethylated HA's, especially in the grains-legumes rotation. The methylated HA's contained more C and H than did the unmethylated ones. Methylation increased the OCH 3 content from 1.52 to 2.28% to between 16.65 and 17.65%. TABLE I1 Elementary composition and methoxyl content of HA's (%; dry-ash free basis) Unmethylated HA C
H
Methylated HA OCH s
C
H
OCH s
Five-year rotation of grains and legumes: Check
53.4
4.9
1.5
58.0
5.9
17.5
Manure
53.7
5.0
2.3
57.5
5.9
17.6
NPKS
54.9
5.1
1.6
57.7
6.3
17.1
Lime
57.4
4.8
1.8
59.9
6.6
17.0
Wheat-fallow sequence: Check
54.4
4.6
1.8
58.8
6.8
16.7
Manure
55.4
4.0
2.2
59.7
6.0
17.5
NPKS
55.7
4.5
1.7
59.3
6.2
17.6
Lime
56.1
4.4
1.6
59.6
6.9
16.7
Separation and identification of oxMation products The gas chromatographic separation of the products resulting from the oxidation of methylated HA extracted from the check sample of the grains and legumes rotation is illustrated in Fig. 1. Gas chromatographic separations o f the HA's extracted from the other soil samples were qualitatively similar to that shown in Fig.1. The results are summarized in Table III. The upper part o f the table lists fully methylated benzenecarboxylic acids,
PERMANGANATE OXIDATION OF HUMIC ACIDS
117
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118
S.U. KHAN AND M. SCHNITZER
the lower part, fully methylated phenolic acids. In addition to the compounds shown, we also isolated and identified small amounts of dimethyl esters of glutaric, adipic, pimelic, 1,2,3-propanetricarboxylic, suberic, azelaic and 1,2,3,4-butane-tetracarboxylic acids. In view of the relatively low OCH3-content of the unmethylated HA's (Table II), it is likely that most COOH and phenolic OH groups were methylated in the laboratory, so that it may be more realistic to refer to the oxidation products as benzenecarboxylic and phenolic acids rather than as esters and ethers.
Effects o f cultural and fertilizer treatments on compounds isolated The permanganate oxidation of all HA's yielded two types of major products (Table llI): (a) benzenecarboxylic acids and (b) phenolic acids. Chemical structures of the compounds that we isolated and were able to identify are listed in Fig.2.
1.
al--R2=C02CH 9 , R3=a4=RS=Wo=H
2. al=a3=co2cH 3 , R2=a~=~=ae--a
R6 i~
a2 a3
9.
Rl=a~=coaca3 , R2=rt3=as=a6=a
4. 5.
rk--a2=a3~02ca3 , a4--as=a6=H rtl=~--r~z=co2cR3 , %=aS--a6=a
6. al=rt3=aS--coaea3 , a2=rtZ=a6~ 7.
RI=R3=R5=CO2CH3 , R2=O~H3 , R4=R6=H
s.
al=~=a3=coacH3 , aS=oea3 , a4=a6=a
J.o.
~l=a2--a3=a4=co2ca3 , aS--a6=a
1.!.. ~=az=R4=rts=coacn3 , •=a6=H 12. al=R2=R3=a5---C02CH 3 , R4=%=H 13. al=rtz=a3=a4=cO2CH3 , ~=0CH3 , a6=H 1,~. 16.
RI=R2=R3=R4=R5=C02CH3 , R6=H RI=R2=R3=R4=R5:=CO2Cti3 , R6=0CH3
17.
r~l=~--a3 =R4=aS=ae=CO2CH9
co2c~3
c02c~3
i8.
ecH3
oc~3
Fig.2. Chemical structures of compounds identified.
The HA's extracted from the soils subjected to the 5-year rotation of grains and legumes produced, per unit weight, more benzenecarboxylic acids than did those originating from the wheat-fallow sequence (Table III). In the former, benzenecarboxylic acids accounted
PERMANGANATE OXIDATIONOF HUMICACIDS
119
for between about 65 and 69% of the total yield of oxidation products, while in the latter these acids constituted between approximately 55 and 60% of the total yield. By contrast, the type of cropping system did not seem to affect the yields of phenolic acids, which averaged about 20% of the total products (Table III). The most abundant benzenecarboxylic acids isolated from HA's resulting from both cropping systems were 15, 10 and 5. The grains-legumes rotation was more favourable for the synthesis of structures yielding these compounds on oxidation than was the wheat-fallow sequence. A comparison of the data for the check plots with those receiving different fertilizer treatments indicates that the latter reduced the chemical complexity of the HA's if we accept, as has been reported (Randall et al., 1938), that benzenecarboxylic acids usually arise from the oxidation of more complex chemical structures. The most effective treatments for this purpose seemed to be the application of lime. Fertilizer treatments were less effective in lowering the yields of phenolic than of benzenecarboxylic acids. Again, lime was most efficient in lowering the proportion of HA structures yielding phenolic acids on oxidation. In the case of the wheat-fallow sequence, the application of manure had a similar but less pronounced effect. Other useful indexes of the relative molecular complexities of the HA's extracted from soils subjected to the different treatmentswere the total yields of oxidation products. These, regardless of cropping system, were highest for the check plots, considerably lower for the manure treatments and lowest where lime was applied. Especially interesting is the isolation of compound 18, which was the only biphenyl compound that we were able to identify and which most likely originated from lignin (Freudenberg et al., 1962). It is especially noteworthy that by means of the analytical techniques that we have employed we were able to identify between approximately 75 and 90% of the oxidation products. We believe that the general approach described in this paper adds a new dimension to soil chemistry by directly relating the effects of cropping systems and fertilizer treatments to the "cores", that is, the more resistant chemical structures of HA's. The data presented herein show that the type of rotation, and especially the application of lime and manure, have significant effects on the synthesis and degradation of these structures. Whether the resulting increased degradation is brought about by enhanced microbiological activity, due to these treatments, and/or by chemical means awaits further investigation. The data reported are in general agreement with those obtained previously (Khan, 1970) which showed that the wheat-fallow sequence as well as lime, and to some extent manure applications, increased the neutralization capacity, especially the COOH group content, of the same HA's. These characteristics may be taken as indexes of increased humification. Thus, a low yield of products resulting from the permanganate oxidation of HA's may be indicative of a relatively high degree of humification, so that permanganate oxidation may serve as a guide for assessing the degree of humification of soil HA's.
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ACKNOWLEDGEMENTS We are i n d e b t e d to the D e p a r t m e n t o f Soil Science, University o f Alberta, for allowing us to use soils f r o m the Breton plots. We are grateful to D. Friessen and J.G. Desjardins for technical assistance. REFERENCES Freudenberg, K., Chen-Loung, C. and Cardinale, G., 1962. Die Oxydation des methylierten and naturlichen Lignins. Chem. Ber., 95: 2814-2828. Freudenberg, K. and Neish, A.C., 1968. Constitution and Biosynthesis of Lignin. Springer Verlag, New York, N.Y., 79 pp. Khan, S.U., 1969. Some carbohydrate fractions of a gray wooded soil as influenced by cropping systems and fertilizers. Can. J. Soil. SCL, 49: 219-224. Khan, S.U., 1970. Enzymatic activity in a gray wooded soil as influenced by cropping systems and fertilizers. SoilBiol. Biochem., 2: 137-139. Khan, S.U., 1970. Humic acid fraction of a Gray Wooded Soil as influenced by cropping systems and fertilizers. Geoderma, 3: 247-254. Khan, S.U. and Schnitzel M., 1971. Further investigations on the chemistry of fulvic acid, a soil humic fraction. Can. J. Chem., 49: 2302-2309. OgneL G. and Schnitzer, M., 1971. The chemistry of fulvic acid, a soil humic fraction, and its relation to lignin. Can. J. Chem., 49: 1053-1063. Pearl, I.A., 1967. The Chemistry ofLignin. Dekker Inc., New York, N.Y., 199 pp. Randall, R.B., Benger, M. and Groocock, C.M., 1938. The alkaline permanganate oxidation of organic substances selected for their bearing upon the chemical constitution of coal. Proc. Roy Soc. Lond., Ser. A, 165: 432-452. Schnitzer, M. and Desjardins, J.G., 1970. Alkaline permanganate oxidation of methylated and unmethylated fulvic acid. SoilSci. Soc. Am. Proc., 34: 77-79. Toogood, J.A., and Lynch, D.L., 1959. Effect of cropping systems and fertilizers on mean weightdiameter of aggregates of Breton plot soils. Can. J. Soil ScL, 39: 151-156. Toogood, J.A., Bentley, C.F., Webster, G.R. and Moore, A.W., 1962. Gray Wooded Soils and Their Management. Univ. of Alberta, Edmonton, Alta., 21 pp.