Microbiological Studies on the Humification Process (Part 2) on the Aerobic Decomposition of the Fresh Plant Residue in the Case of Single Inoculation

The humification of several constituents of plant was studied by Trussow¹⁾ and Waksmans²⁾ in the Past, or by Kononova,³⁾ Tepper⁴⁾ and Laatsch⁵⁾ in recent years. In the previous paper, we experimented the microbial action on humus separated from an uncultivated soil, and showed that the inoculation of Trichoderma koningi and soil extract prqmoted the humification of the sample, but such tendency was not revealed in the case of single inocolation of two strains of Actinomyces and four strains of aerobic bacteria, and that when the humification of the soil humus was promoted it occurred, in the first place, as the transformation of the fulvic acid fraction, not as that of the precipitation part (the humic acid fraction). Following the previous report, we studied the humification process of fresh plant residue (upland rice straw) by a similar procedure.     

Characteristics of Humic Substances in Paddy Soils

In the present paper,the composition of humus and the charateristics of humic acid from seven paddy soils were compared with those of upland (and/or natural) soils.Results show that:(1) in each group of the soil samples for comparison the HA/FA ratio of the humus of a paddy soil,in most cases,was appreciable higher than that of adjacent upland(and/or natural) soil derived from the same parent material;(2) the humic acid extracted from the paddy soils was characterized by a higher C/O ratio,a higher content of methoxyl groups,and a lower content of carboxyl groups than those from the corresponding upland (and/or natural) flooded soils,implying that the humic acid formed under rice cultivation is in a lower degree of humification than that formed under upland(and/or natural) conditions;and (3) the humic acid of paddy soils,however,was not always characterized by a lower aromaticity than that of the corresponding upland(and/or natural) soils.     

Chemical studies on soil humic acids

The levels of alcoholic and phenolic hydroxyl, methoxyl, carboxyl, and carbonyl groups of 33 to 38 humic acids obtained from various types of soils were determined to analyze the relationships between the amounts of these functional groups and the degree of humification or the types of soils. The amounts of various oxygen-containing functional groups examined were all proven to be significantly different among the various types of humic acids by analyses of variance. During humification. generally, the carboxyl and carbonyl groups increased while alcoholic and phenolic hydroxyl and methoxyl groups decreased. Linear and logarithmic regression analyses of carboxyl group contents on RF values (optical density of the alkaline solution of humic acids at 600 nm) gave very significant positive correlations. The carboxyl group contents of Rp type humic acids and humic acids from calcareous soils were largely distributed in the upper side of the regression curve. The carbonyl group contents showed a very significant linear positive correlation with carboxyl group contents. and both of them showed high linear positive correlation with RF values. Phenolic hydroxyl group contents decreased with humification in Rp(l). B. and A type humic acids. Alcoholic hydroxyl group contents showed a significant negative linear association with _RF values. Methoxyl group contents decreased rapidly with increasing humification in low humified humic acids, and their negative correlation with _RF values were proven to be very significant by logarithmic regression analysis. The complicated relationship between oxygen content and RF value which was reported previously (7) has been accounted for by the results obtained in the experiments conducted here.     

长期施肥条件下土壤养分的动态和平衡——Ⅰ对土壤腐殖质积累及其品质的影响

长期施用有机肥和化肥,对土壤腐殖质积累和改善品质的作用研究,于1986～1991在俄罗斯季米里亚捷夫农学院等地进行。主要结果如下:.1.有机肥对土壤腐殖质的积累作用大于NPK化肥。施肥所增加的土壤腐殖质,除了砂壤土外,主要是在0～60cm土层,60cm以下土层施肥效果不明显。.2.长期施用有机肥和NPK化肥,提高了胡敏酸组分Ⅰ(游离的及与活性R₂O₃结合态)的含量和腐殖酸相对迁移率,而与Ca⁺⁺离子结合的胡敏酸组分Ⅱ含量下降。由于胡敏酸组分Ⅱ与Ca⁺⁺离子结合减弱,增加了腐殖质在水中的溶解,使之在大雨或大量灌水时易于淋失。.3.施用有机肥和NPK化肥,水溶性腐殖物质的含量明显提高,缓解了干旱条件下土壤高浓度矿物盐的影响。.4.长期施肥提高了土壤的亲水性;其膨胀容积、膨胀速度常数、土壤吸水量、土壤表面积等指标均不同程度的提高,但砂壤土增加了幅度低于粉砂壤土和粘壤土。.5.胡敏酸甲氧基功能团的含量多寡是衡量土壤腐殖质化的重要指标。试验表明,长期施肥下低肥力酸性土壤中胡敏酸甲氧基含量提高了46％～300％,高肥力非酸性土壤提高了17％～32％。     

Effect of organic amendment on amount and chemical characteristics of humic acids in upland field soils

To assess the effect of continuous organic material (OM) application on soil humic acids, the amount and chemical characteristics of humic acids in various types of soils (n = 10) were compared between plots treated with farmyard manure (FYM) or rice straw compost (RSC) plus chemical fertilizer (CF) and plots treated with CF alone. The degree of humification (degree of darkening), molecular size distribution and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance spectra of humic acids from CF‐treated soils showed wide variation among the soils. Humic acid content was generally larger in OM + CF soils than in corresponding CF soils, and the stable C isotopic ratio suggested partial replacement of indigenous humic acids with OM‐derived ones even where no apparent increase in humic acid content was observed. The rate of OM application and the indigenous humic acid content were related positively and negatively, respectively, to the apparent accumulation rate of humic acids among soils. The degree of humification of humic acids was generally smaller in OM + CF soils than in CF soils. Humic acids extracted from FYM and RSC exhibited chemical characteristics typical of humic acids having a smaller degree of humification, which suggested the contribution of OM‐derived humic acids to the differences between OM + CF and CF soil humic acids, such as larger average molecular sizes and smaller and larger proportions of aromatic C and O‐alkyl C, respectively, relative to total C in the OM + CF soil humic acids. Little change was observed in the chemical characteristics of humic acids when the degree of humification of indigenous humic acids was small. The effect of OM application on the chemical characteristics of humic acids was most conspicuous in soils containing humic acids having an intermediate degree of humification, possibly resulting from the combination of accelerated degradation of indigenous humic acids and the accumulation of OM‐derived humic acids.     

Characterisation of a humus profile under beech forest by using wet-chemistry and CPMAS 13C NMR-spectroscopy in consideration of spatial heterogeneity

Three of ten macromorphologically similar humus profiles of a Dystric Cambisol under a beech forest were randomly chosen and the litter and humus layers were characterized by wet-chemistry and CPMAS ¹³C NMR-spectroscopy. The spatial heterogeneity of the litter and humus layers was high for the bulk density and for the sugar and starch fraction; lowest coefficients of variation were obtained for the chemical shift range of O-Alkyl-C. The spatial heterogeneity of the layers did not differ by means of all chemical methods. Changes in the chemical composition at the beginning of humification were high from the fresh fallen litter (Ln-layer) to the morphologically only slightly altered litter in the L-layer, whereas the changes in the litter and humus layers from the L to the Ohf-layer were minor. However, the organic matter of the first mineral horizon was characterized by a decrease in lignin and cellulose. With wet-chemistry and ¹³C NMR-spectroscopy similar results were obtained for polysaccharides whereas results which can be attributed to changes in lignin deduced by the methoxyl content differed from each other.     

Several properties of humic acids

In the preceding paper¹), it was shown that the elementary composition of soil humic acid changed regularly with the progress of humification. Afterwards, the author conducted several experiments on the base exchange capacity, the content of hydrolysable nitrogen, the resistance to oxidizing reagent, the electrolyte coagulation and X-ray analysis of various humic acids. These experimental resu1ts will be given in this report.     

Analysis of polynuclear aromatic and aliphatic components in soil humic acids using ruthenium tetroxide oxidation

Although condensed aromatic components are considered to be one of the major structural units of soil humic acids (HAs) and to be responsible for the dark colour of HAs, their amount and composition remain largely unknown. In ruthenium tetroxide oxidation (RTO), condensed aromatic components are detectable as their degradation products, mainly benzenepolycarboxylic acids (BPCAs). We applied this technique to soil HAs with various degrees of humification (darkening). The yields of water‐ and dichloromethane‐soluble products from HAs upon RTO after methylation ranged from 210 to 430 mg g⁻¹ and 10–40 mg g⁻¹, respectively. Eight kinds of BPCAs with two to six carboxyl groups, and seven kinds of BPCAs with additional side chains (tentative assignment) were obtained as methylated counterparts. The yield of each BPCA and the sum of the yields of BPCAs (12–85 mg g⁻¹ HAs) increased with increasing degree of humification and aromatic C content. The compositions of BPCAs indicated that the degree of condensation was greater in the HAs with greater degrees of humification. The sum of the yields of aliphatic compounds ranged from 0.1 to 6.5 mg g⁻¹, and decreased with increasing degree of humification. The C₁₂ to C₃₀ monocarboxylic acid methyl esters accounted for > 56% of the aliphatic compounds assigned, which may be present mainly as end alkyl groups in the HA molecules. We also obtained the methylated counterparts of C₁₄ to C₂₄ dicarboxylic acids; these were possibly derived from polymethylene bridges between adjacent aromatic rings.     

Carbon and Nitrogen Mineralization and Humus Composition Following Municipal Solid Waste Compost Addition to Laterite Soils under Continuous Cassava Cultivation

A glasshouse incubation experiment was conducted to study the carbon (C) and nitrogen (N) mineralization of municipal solid waste compost (MSWC) added at differential rates to a laterite soil where cassava has been continuously cultivated for the past 10 years. The rate of C mineralization from added substrates increased with increasing rates of addition of MSWC. Available N significantly increased with increase in the rate of application of MSWC. There was a decreasing trend in E₄₆₅/E₆₆₅ ratio of humic acid as we increased the rate of application of MSWC from 2.5 to 20 t ha^?1. The Cross Polarization Magic Angle Spinning (CPMAS) ¹³C NMR spectral analysis revealed that there are differences in the rate of humification of added MSWC, and application of MSWC at 15 t ha^?1 resulted in least humification with the greatest alkyl C, lowest aromatic C, and greater O-alkyl C content. The decomposition rate (R) was found to be greater for this treatment. The residual C in soil was found to increase over time coincident with greater rates of MSWC application, indicating increased C stabilization, which could improve soil quality.     

Novel method for the determination of the methoxyl content in lignin by headspace gas chromatography

The paper reports on a headspace gas chromatographic (HS-GC) method for the determination of methoxyl in lignin. The method involves the quantitive cleavage of methoxyl with hydroiodic acid (HI) to form methyl iodide in a closed headspace sample vial at 130 °C for 30 min. After HI has been added, the sample is neutralized by injecting a sodium hydroxide solution; the methyl iodide in the vial was determined by HS-GC using a flame ionization detector. The results showed that the method has an excellent measurement precision (RSD < 0.69%) and accuracy (RSD < 3.5%) for the quantification of methoxyl content in lignin. The present method is simple and accurate and can be used for the efficient determination of methoxy1 content in lignin and related materials.     

Biological,Physicochemical, and Spectral Properties of Aerated Compost Extracts: Influence of Aeration Quantity

The goal of this experiment was to investigate the effect of aeration quantity (0, 11, 33, 55, and 77 L·min^?1) on the growth of aerated compost extracts from a pig manure–straw compost. When the aeration quantity was 11 L·min^?1, lettuce root growth enhancement of normalized compost extracts was at a maximum. As the aeration quantity increased, the total water-soluble organic carbon (TWSOC), total nitrogen (TN), total phosphorus (TP), humic carbon (humic C) content, and humification degree of compost extracts improved gradually. No differences in functional group structure were found among the aerated compost extracts. The positive root growth could be attributed to physicochemical and spectral characteristics, such as TN content, humic substances content, humification, aromaticity, and the low content of carboxyl groups. In conclusion, the aeration quantity of 11 L·min^?1 was suitable for the production of aerated compost extracts, which obtained much greater promotion growth.     

13C solid-state NMR assessment of decomposition pattern during co-composting of sewage sludge and green wastes

The fate of organic matter during composting is poorly understood. Therefore, we analysed composts of sewage sludges and green wastes (44 samples representative of 11 stages of biodegradation) by conventional chemical methods: pH, humic (HA) and fulvic acid (FA) content, C, N and organic matter (OM) content, and by ¹³C CPMAS NMR to assess the decomposition process of the organic matter. Chemical changes clearly occurred in two phases: first, decomposition of OM during the first 2 months was characterized by decreased C/N ratios, OM content and increased pH; and second, a humification process with increased HA/FA ratios. NMR spectrum changes confirmed this pattern, with an increase in aromaticity and a decrease in alkyl C. A decrease of syringyl to guaiacyl ratio (S/G), a sign of lignin transformation, also indicated humification during composting. NMR spectroscopic properties of composts were also studied by means of principal components analysis (PCA) and revealed changes according to the degree of compost maturation. The factorial map presents a chronological distribution of composts on the two first principal components. The influences of eight chemical factors on the PCA ordination of composts as monitored by their evolution by NMR were also studied by multivariate analyses. PCA clearly indicated two phases: the rapid decomposition of organic matter followed by the formation of humic‐like substances. The first phase, that is ‘new’ composts, was strongly correlated with OM contents, pH and C/N ratios whereas the second phase, corresponding to ‘old’ compost, was correlated with pH, HA content and HA/FA ratio. These results confirm that knowledge of the formation of humic substances is indispensable to suitable monitoring of the composting process.     

Total and carboxyl acidity and methoxyl groups of humic acid preparations of chernozemic soils

Abstract

Alkaline‐soluble, acid‐precipitable organic matter from the Ah and Bm horizons of Chernozemic soils developed on four parent materials in each of three soil zones was analyzed for total and carboxyl acidity, and methoxyl groups. The values are expressed as meq/g dry ash‐free organic matter.

Total acidity generally was higher in the Black Chernozems than in the Brown Chernozems and higher in the Bm horizon than in the Ah horizon. The distribution of carboxyl acidity between the Ah and companion Bm horizons of individual soils appeared to be related to texture and rainfall.

The. methoxyl group content of the Brown Chernozems was larger than that of the .Dark Brown Chernozems, which in turn had a larger methoxyl group content than that of the Black Chernozems. It was concluded that the organic matter in the Ah horizons of the Brown Chernozems was not as humified as that of the Black Chernozems. The pH value and a minimum clay content are possible determining factors as to the amounts of methoxyl carbon present.     

Temporal changes in humic acids in cultivated soils with continuous manure application

Abstract

Although the application of manure to upland fields is believed to induce changes in the quality of humic substances in soil as well as the quantity, the direction and extent of these changes have not been elucidated. To understand temporal variations in humic acids, periodically collected soil samples from two fields, a Typic Hapludult (Togo) and a Pachic Melanudand (Kuriyagawa), with cattle manure and chemical fertilizer (CF) were examined. The content and degree of humification (darkening) of the humic acids were distinctly greater in Kuriyagawa than in Togo soil. Corresponding to the difference in the degree of humification, molecular size distribution, elemental composition, infrared (IR) spectra, and ¹³C cross polarization/magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectra of humic acids differed between the two soils. Manure application at 40 Mg ha^?1 year^?1 for 16 years (Togo) and at 80 or 160 Mg ha^?1 year^?1 for 19 years (Kuriyagawa) resulted in greater humic acid content compared with plots with CF only because of its increase in the manured plots and/or decrease in the CF plots. Manure application at an extremely high rate (160 Mg ha^?1 year^?1) resulted in higher H content and greater signal intensities of alkyl C, O-alkyl C and amide C=O in the ¹³C CPMAS NMR and/or IR spectra. Although humic acids with larger molecule sizes increased in all the manured plots, differences between the humic acids from the plots with and without manure applied at practical levels in the elemental and spectroscopic analyses were small or scarce. These results were considered to be because of the similarity between the indigenous soil humic acids and the manure-derived ones in Togo soil (a low degree of humification) and because of the abundance of highly-humified humic acids in Kuriyagawa soil.     

菌剂对鸡粪堆肥腐殖质含量品质的影响

腐殖质是评价堆肥品质的重要因素,该文利用鸡粪和秸秆为原料进行高温好氧堆肥,设计接种菌剂和不接种菌剂(对照)2个处理,研究菌剂添加对堆肥腐殖质形态、含量、品质的影响。结果表明:与对照相比,接种菌剂可以加快有机物的降解,矿质化时间缩短14d,菌剂具有良好的保碳效果,总有机碳含量提高了16.1%,同时总腐殖酸、游离腐殖酸以及水溶态腐殖酸及胡敏酸的含量,分别提高了38.7%,45.7%、39.0%及54.9%。接种菌剂可以提高腐殖酸的活性,堆肥结束后,接种菌剂处理的游离腐殖酸和水溶性腐殖酸含量均增加,而对照处理的含量均降低;堆肥可以提高腐殖酸质量,堆肥结束后两个处理总腐殖酸含量均下降但是缩合度、腐殖化率、腐殖化指数及胡敏酸百分比均提高,特别是添加菌剂的处理腐殖化程度明显高于对照。说明了菌剂可以增加堆肥腐殖质含量,提高腐殖质缩合度、芳构化程度及活性。     

The Influence of Humification Degree of Humic Acid on Its Sorption of Norfloxacin During Sewage Sludge Composting

Norfloxacin (NOR), an antibiotic widely used in livestock and poultry production, has become ubiquitous in the aquatic and terrestrial ecosystems as a result of veterinary excretion of the parent compound or its active metabolites. The sorption of NOR onto humic acid (HA) may influence the fate of NOR in the environment. In the present study, HA was extracted from sewage sludge in different composting stages of days 0, 10, 30, and 70 to investigate the sorption of NOR onto HA as affected by the humification degree of HA. The results of elemental and Fourier transform infrared (FTIR) spectrum analyses showed that the contents of aromatic and carboxylic groups in HA increased with composting time, indicating an increase of humification degree. The result of sorption experiments demonstrated that the HA had a high sorption capacity for NOR with strong nonlinearity. A two-stage sorption was observed in the sorption process with an equilibration time of 48 h. Both the Freundlich model (Adj. R² range 0.988–0.994) and Langmuir model (Adj. R ² range 0.917–0.928) fitted well with all sorption isotherms of the HA samples of different humification degrees. Moreover, the increase of sorption distribution coefficient (K _d ) value with composting time indicated that the sorption affinity of HA for NOR increased with increasing humification degree of HA. The major sorption mechanism was the interaction between NOR and rich aromatic moieties and carboxylic group in the HA.     

Chemical composition of the organic matter in forest soils: The humus layer

Decomposition and humification were studied within three types of forest humus (mull, moder, and mor) by means of CPMAS ¹³C NMR spectroscopy combined with degradative methods. The NMR data show that O-alkyl carbon decreases in all soils, and alkyl as well as carboxyl carbon increase as depth and decomposition increase; the percentage of aromatic carbon remains constant at about 25%. With increasing depth the amount of carbon that can be identified as belonging to specific compound classes by wet chemical methods decreases from 60% to 40%. Microbial polysaccharides and the proportion of non polysaccharide O-alkyl carbon increase with depth. A selective preservation of recalcitrant, condensed lignin structural units is also observed. In order to relate the spectroscopic and chemical data from investigations of whole soils with studies of humification, samples were fractionated into fulvic acid, humic acid, and humin fractions. The fulvic acid fraction contains large concentrations of carbohydrates irrespective of the soil horizon. The humic acid fraction contains less polysaccharides, but high amounts of alkyl carbon and aromatic structures. The percentage of aromatic carbon existing in the humic acid fraction increases with depth, probably reflecting the amount and degree of oxidative decomposition of lignin. A loss of methoxyl and phenolic groups is evident in the ¹³C NMR spectra of the humic acid fraction. The humin fraction resembles relatively unchanged plant-derived materials as evident from the lignin parameters and carbohydrate contents. All the observed data seem to indicate that humic acids originate form oxidative degradation of humin or plant litter.     

The response of soil organic matter to manure amendments in a long-term experiment at Ultuna, Sweden

In a long-term field experiment started in 1956 on a clay loam soil at Uppsala, Sweden, changes of organic carbon in the topsoils receiving various organic amendments at the rate of 200 kg C ha^'1 year^'1 were studied to determine soil organic matter characteristics, variations of δ¹³C in the soil and to estimate a carbon balance. Fallow and mineral fertilizer without N led to a significant decrease of soil organic matter (SOM) in the soil, green manure maintained the SOM content, and animal manure and peat increased the SOM content significantly. The stable portion of the added organic materials after 37 years of continuous input was 12·8, 27·3, and 56·7%, for green manure, animal manure and peat, respectively. This was reflected by half-lives of organic carbon originating from the amendments between 3·0 (green manure) and 14·6 years (peat). The isotopic composition of SOM changed both due to mineralization (continuous fallow) and the addition of amendments is topically different from soil humus (green manure, animal manure). The isotopic effect was used to calculate the percentage of carbon derived from animal manure present for the year 1993. This value (55·4%) was larger than that derived from the carbon balance, which indicated a priming effect of the animal manure on the initial soil humus. Mineralization of microbially available organic substances led to an increase in the degree of humification on plots not receiving organic amendments. Adding peat and animal manure resulted in a decrease of the humification index due to the continuous input of poorly humified material. The extinction ratio (E₄/E₆) and ratio of fulvic acid to humic acid changed considerably in the peat treated plots. Fourier transform infrared (FTIR)-measurements of the extracts showed that peat characteristics can be detected in peat treated soils. The other amendments did not alter the characteristics of the extractable humic substances.     

Soil fulvic acid properties as a means to assess the use of pig slurry amendment

Soil amendment with animal manures is a common practice for either increasing soil organic matter (SOM) and nutrient content or disposing of wastes from intensive animal industries. However, the application of organic amendments that are not sufficiently mature and stable may adversely affect soil properties, especially the content and quality of SOM pools. In this work, the effect of the consecutive annual additions of pig slurry (PS) at rates of 0 (control), 90 and 150 m³ ha⁻¹ per year over a 4-year period on the soil fulvic acid (FA) fraction of SOM was investigated in a field plot experiment conducted under semiarid conditions on a Calcic Luvisol in Toledo province, Spain. The FAs isolated from PS and control and amended soils were characterized for chemical, compositional, structural and functional properties by use of elemental and functional group analysis, and ultraviolet/visible, Fourier transform infrared (FT IR), fluorescence and electron spin resonance (ESR) spectroscopies. PS-FA was characterized by a prevalent aliphatic character, large contents of acidic functional groups, S- and N-containing groups and polysaccharide components, extended molecular heterogeneity, small organic free radical (OFR) contents and small degrees of aromatic ring polycondensation, polymerization and humification. With respect to the control soil FA, the PS-amended soil FAs were characterized by a smaller extraction yield, O and OFR contents and ratios of absorbances at 465 and 665 nm, and larger C, N, S, COOH and phenolic OH contents, C/N ratios and aliphaticity. Statistical analysis of experimental data showed that, with some exceptions, these effects generally increased with increasing cumulative amount of PS applied to soil over time. In conclusion, cumulative PS application to soil over time modifies the content and properties of the FA fraction of SOM. Thus, this material should not be considered as a mature organic amendment and should be treated appropriately before it is applied to soil, so as to increase the degree of humification and enhance its potential as a soil organic fertilizer.     

Accumulation of heterocyclic nitrogen in humified organic matter: a 15N‐NMR study of lowland rice soils

Recent intensification of cropping and the attendant longer submergence of the soil for lowland rice in tropical Asia appear to have altered the nature of the soil organic matter, and perhaps also nutrient cycling. To identify the dominant forms of organic nitrogen in the soils we extracted the labile mobile humic acid (MHA) and the more recalcitrant calcium humate (CaHA) fractions from soils under several long‐term field experiments in the Philippines and analysed them by ¹⁵N‐nuclear magnetic resonance spectroscopy. Amide N dominated the spectra of all humic acid (HA) samples (60–80% of total peak area). Its proportion of total spectral area increased with increasing intensity of cropping and length of time during which the soil was flooded and was greater in the MHA fraction than in the CaHA fraction. Simultaneously the spectral proportion of free amino N and other chemical shift regions decreased slightly with increasing length of submergence. Heterocyclic N was detected at modest proportions (7–22%) and was more prevalent in more humified samples, especially in the CaHA of aerated soils. Correlations of spectral proportions of heterocyclic N with other properties of the HA, reported elsewhere, were highly significant. Correlations were positive with visible light absorption (r= 0.86) and concentration of free radicals (r= 0.85), both of which are indices of humification, and negative with concentration of H (r= ?0.86), a negative index of humification. Correlations of spectral proportions of amide N with these properties were also highly significant but in each case of opposite sign to that of heterocyclic N. Proportions of heterocyclic N declined with increasing duration of submergence. The results suggest that (i) ¹⁵N‐NMR can reproducibly measure some portion of heterocyclic N, (ii) formation of heterocyclic N is associated solely with gradual humification occurring over many years, and (iii) the abundant phenols in the submerged rice soils did not promote formation of heterocyclic N, and hence some other process is responsible for a substantial decrease in the availability of native N associated with intensive rice cropping.