Characterization of fungal melanins and soil humic acids by chemical analysis and infrared spectroscopy

Summary Humic acids from two Brazilian topsoils under savanna grassland and five soil fungal melanins were characterized by elemental, functional group and infrared analysis. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for several other fungal melanins and soil humic acids. Compared with the soil humic acids, the infrared spectra of the fungal melanins showed greater detail, indicative of higher aliphaticity. They were similar to the type III infrared spectra of humic acids, which are characteristically high in proteinaceous material and polysaccharides. The infrared spectra of the humic acids from the two Brazilian soils studied were classified as type I, which includes most soil humic acids. Notwithstanding the greater detail, in some areas the fungal melanin spectra were similar to those reported for other fungal melanins and humic acids of different origins. The probable contribution of the melanic fungi to the formation of soil humic polymers is discussed.     

Comparisons of IR-spectra from melanins of microscopic soil fungi,humic acids and model phenol polymers

Microscopic soil fungi, such as Epicoccum nigrum, Stachybotrys chartarum, Hendersonula toruloidea and others when cultivated on glucose form high molecular weight melanins in the culture media and in the cells. The melanins resemble humic acids with respect to certain chemical properties and the IR-spectra also indicate similarities in functional groups. However, bands at 2900, 1460 and 1380 cm^?1 suggest a higher content of aliphatic CH-components, and a pronounced shoulder at 1540 cm^?1 suggests peptide-like material. A shoulder at 1720 cm^?1 and a broad band at 1630–1650 cm^?1 form evidence of fewer free carboxyl groups than in most soil humic-acid preparations. Upon hydrolysis with 6N HCl methylation or a short heat treatment, distinct adsorptions at 1720 and in the 1600 cm^?1 region appeared in the residues, indicating C=O vibrations of free carboxyl groups and aromatic C=C vibration. Comparisons with the variability of the carboxyl band in model polymers from phenols of various structures and prepared either by phenolase or autoxidative polymerization in the presence or absence of amino-acid compounds, suggested that the carboxyl group in fungal melanins may be bonded by H-bridges which shift the C=O absorption into lower frequences.Addition of clay minerals to the culture media of fungi accelerated or increased biomass and melanin formation. Comparison of band locations and shape of the spectra from the melanins with or without clays indicated that no basic alteration occurred in the composition of the melanins from clay culture. However, a higher ash content and the presence of specific bands from the added clays, together with sharpening and a shifting of the broad 1650 cm^?1 band from the controls into higher frequencies, suggest chemical bonding of the melanins with clays through participation of the carboxyl groups. The model polymers from specific mixtures of phenols together with peptones and prepared either by oxidation with phenolase or by autoxidation, closely resembled the fungal melanins. This represents strong evidence for a similarity in the structural units and their linkages in both the melanins and the model polymers. The similarity of fungal melanins to some humic acids from recent lake and marine sediments and soil humic-acid fractions is discussed.     

Elemental,functional group and infrared spectroscopic analysis of actinomycete melanins from brazilian soils

Eleven actinomycete melanins were characterized by elemental and functional group and infrared analysis. A soil humic acid from a Brazilian topsoil, a darkred latosol under savanna grassland, analysed previously, was used for comparative purposes. C, N, total acidity, COOH, and phenolic OH contents were within the ranges reported for soil humic acids and fungal melanins. Compared to the soil humic acid, the actinomycete melanins showed greater detail, indicative of higher aliphaticity. Most of these were, in varying degrees, similar to the type III IR spectra of humic acids, which are characteristically high in proteinaceous material, and with variable amounts of polysaccharides. The exceptions were two melanin spectra that showed more resemblance to the humic acid from the dark-red latosol, which belongs to the type I spectra of soil humic acids, a category that includes most soil humic acids. The probable participation of melanic actinomycetes in the formation of humic polymers in discussed.     

Elemental,functional infrared and free radical characterization of humic acid-type fungal polymers (melanins)

Summary Humic acid-type polymers (melanins) synthesized in culture media by the fungi Aspergillus glaucus, Eurotium echinulatum, Hendersonula toruloidea, Stachybotrys atra and Aspergillus sydowi were analysed for elemental composition, functional group content, infrared (IR) and electron spin resonance (ESR) properties. Results were discussed in comparison with range values referred for soil humic acids. The fungal polymers showed significant differences in carboxyl and nitrogen content and C/H atomic ratios, reflecting a different degree of condensation (aromaticity) among the various samples. IR analysis gave evidence of: (a) the predominant aromatic character of melanins from A. glaucus, E. echinulatum and H. toruloidea; (b) the high content of aliphatic and olefinic components of S. atra melanin; (c) the typical presence of amide bonds in the nitrogen-richest melanins from A. sydowi and H. toruloidea; and (d) the generally low amount of free carboxyl groups, which often appeared involved in hydrogen bonds. ESR spectra showed that all the melanins studied contained appreciable concentrations of organic free radicals of prevailing semiquinonic nature and of the same order of magnitude commonly measured in humic acids from soil and other sources. The free electron concentration was shown to be directly related to the C/H atomic ratio and to the degree of aromaticity shown by IR analysis. This indicated that the highest free radical content in the melanins from E. echinulatum and A. glaucus was associated with the highest presence of condensed aromatic structures. Humic acid-type polymers synthesized by soil fungi may, therefore, contribute to the total free radical content of soil humic substances and play important roles in all reactions involving free radicals in soils and related environments.     

Low boiling-point compounds produced by pyrolysis of fungal melanins and model phenolic polymers

Pyrolysis-gas chromatography-mass spectrometry was used to characterize fungal melanins and model phenolic polymers. Samples were pyrolysed at 700°C for 10 s and the low boiling-point compounds were separated on a Chromosorb 102 column. Low boiling-point compounds identified with a mass spectrometer coupled to the gas Chromatograph were very similar for peptone, a model autoxidative phenolic polymer incorporating peptone, and some fungal melanins. The major low boiling-point components from fungal melanins were related to proteins and to polysaccharides. A nitrogen-free phenolic polymer yielded very small amounts of low boiling-point pyrolysis fragments. The compounds identified appeared to be derived largely from the peptide, carbohydrate, and possibly small amounts of other aliphatic materials in the polymer structures.     

Further investigations on the chemistry of fungal “humic acids”

In view of the considerable interest in laboratory-prepared fungal “humic acids” as possible precursors or incorporated structural components of soil humic substances, we degraded four fungal “humic acids” by the relatively mild alkaline cupric oxide oxidation. The oxidation products were extracted into organic solvents, methylated, separated by thin-layer chromatography and identified on a gas chromatographic-mass spectrometric-computer system.Average yields of major degradation products were: (a) aliphatic compounds, 38 per cent; (b) benzene-carboxylic compounds, 25 per cent; and (c) phenolic compounds, 21 per cent. The remaining 16 per cent consisted of a number of dialkyl phthalates. Our data agree with those that we reported earlier when we degraded a number of fungal “humic acids” by the more drastic alkaline permanganate oxidation and show that fungal “humic acids” are enormously complex organic materials containing aliphatic and aromatic structures, (some of which contain N), but only a relatively small proportion of which is phenolic. Most of the aliphatics isolated consisted of alkanes and fatty acids, which are known to persist in soils over long periods of time and are frequently firmly retained by soil humic substances.     

Abbau und Umwandlung von Pflanzenrückständen und ihren Inhaltsstoffen durch die Mikroflora des Bodens

Degradation and transformation of plant residues and their components by the microflora of the soil This review describes recent results of publications in this area. It condsiders the dynamics of degradation under field and laboratory conditions and indicates some of the problems of simulation models. The preponderant part of transformation processes apparently takes place in a relatively small fraction of soil organic matter which also includes the biomass. Methods for the quantitative measurements of the biomass have been considerably improved. The turnover rates of plant residues are effectively influenced by their chemical composition, for instance by their C/N ratio and their content of lignin or polysaccharides. C/N ratios also seem to influence the socalled priming effect and the transformation of increasing amounts of plant residues added to the soil. Some progress has been also made in the transformation of plant, residues under different climatic conditions. Experiments with polysaccharides and glucose have indicated that a major portion of the residual carbon residues in soil are contained in the biomass or N-containing microbial metabolites. A small part is also present in the phenolic constituents of humic compounds. Root excretions also contribute carbohydrates. Recent experiments indicated the transportation of a considerable amount of photosynthetic products from sprouts through roots into the soil. They cause intensive microbial turnover processes in the root zone. N-containing compounds are stabilized in the soil biomass. Proteins and aminopolysaccharides are furthermore stabilized by sorption on humic compounds or clay particles. They are even more effectively stabilized by linkage into humic compounds. Plant residues contain appreciable amounts of free and polymerized phenols which are degraded by microorganisms or incorporated into humic compounds. Easily oxidisable phenols are more slowly degraded in soil than the more stable ones. This is especially true for low concentrations of phenols added to the soil. The pH-values of the soil and its content of humic compounds influence essentially the degradation rate. Lignins belong to the most important natural biopolymers. Methods for their specific labelling with 14°C have essentially promoted studies about their degradation and transformation in soil and have enlarged the knowledge about lignin degrading organisms. Beside lignins and other plant polyphenols, melanins may play an important role in the formation of humic compounds. These melanins are synthesized form carbohydrates by some microscopic fungi through secondary metabolic reactions. They are formed from phenols, quinones, polyenes, aminoacid and aminosugar derivatives. Similar to humic acids they are relatively stabile against rapid microbial degradation. Analytical methods developed in the last years indicate that both groups of polymers contain similar building blocks.     

Effect of fungal humic-like polymers and their phenolic units on auxin destruction

Humic-like substances synthesized by Aspergillus sydowi, Epicoccum nigrum, Hendersonula toruloidea and Stachyhotrys atra inhibited indoleacetic acid (IAA) destruction by lentil roots extracts. The fungus polymers were similar to soil humic acids with respect to the effect on IAA-oxidase activity. Model polymers prepared using pure phenols reduced the IAA oxidation too. The relationships between the effect of fungal substances on IAA-oxidase activity and their phenolic units content are briefly discussed.     

Comparisons of soil organic matter and its fractions by pyrolysis mass-spectrometry

Pyrolysis mass-spectra from a sample of the A₁-horizon of a soil from southern Spain showed predominant peaks related to furan derivatives similar to those observed from complex polysaccharides in which not only hexoses but also pentoses and deoxyhexoses were constituent units. Smaller peaks, typical for protein materials and phenolic units, were also observed. On the other hand, typical peaks for the methoxyphenols of lignins were very small and indicated only limited amounts of undecomposed lignin residues in this soil sample. Peaks related to benzene or toluene were also very small.Humic acid samples from this soil showed much more prominent signals related to protein materials, benzene and phenolic derivatives and weaker polysaccharide-related signals than did the entire sample. Typical lignin related peaks were small or insignificant. Spectra from the grey or brown humidic acid fractions were much like those of the parent humic acid. Brown humic acid, however, showed stronger signals for nitrogen and sulphur compounds, indicating a higher content of protein-like materials in this fraction. Preparations of humic acid hydrolyzed by 6 N HCl showed in their pyrolysis products a marked increase in phenols and methoxyphenols.In its pyrogram, humin resembled humic acid, but signals for complex polysaccharides were more evident. Lignin-like materials seem not to be higher in this fraction. Hymatomelanic acid showed prominent signals related to polysaccharides and lignin. Pyrograms from the soil polysaccharides showed the characteristic pattern of a complex polysaccharide with the presence of fragments from polymers of amino acids or amino sugars. Fulvic acid spectra showed obvious dissimilarities to those from humic acid in that signals for protein, as well as those related to phenols, were low. Depending upon the isolation method, the fulvic acid preparations showed differing signals related to polysaccharide or phenolic materials.     

Chemical–structural information from solid-state 13C NMR studies of a suite of humic materials from a lower montane forest soil,Colorado, USA

Chemically and physically fractionated samples extracted from the surface horizon of a soil developed under a mix of coniferous and deciduous vegetation in southwestern Colorado were studied. ¹³C NMR data on this soil's organic matter and its HF(aq)-washed residue, as well as the classic acid/base-separated humic fractions (humic acid, fulvic acid, humin), were examined for chemical–structural detail, e.g., the various structural functionalities present (especially lipids, carbohydrates, aromatics, polypeptides and carbonyl/carboxyls). Among the humic fractions, it was found that the lipid concentrations are in the order humic acid>fulvic acid= humin; for carbohydrates the order is fulvic acid>humin>humic acid; for aromatic carbons the order is humic acid>humin>fulvic acid; for polypeptides it is humic acid>fulvic acid>humin and for carbonyl/carboxyl species it is humin>humic acid>fulvic acid, but the differences are small. ¹³C spin–lattice relaxation times indicate that at least two types of “domains” exist in each, corresponding to “higher” and “lower” concentrations of paramagnetic centers, e.g., Fe³⁺.     

紫色水稻土胡敏酸的形成——稻草腐解试验

土壤腐植酸类物质的形成是土壤固碳的重要过程,但对腐植酸类物质形成过程的了解仍不甚清楚,为了丰富土壤腐植酸类物质形成理论,采用富立叶变换红外光谱和固态交叉极化-魔角旋转^13C-核磁共振光谱技术分析了紫色水稻土稻草腐解过程中胡敏酸的波谱学特征。结果表明,稻草腐解的前期,胡敏酸的红外光谱所有吸收峰（3364、2933、1653、1599、1508、1461、1421、1331、1225、1126、1033cm^-1）强度皆有明显减弱,核磁共振光谱的烷基、多羟基和芳基的共振峰明显减弱且甲氧基的共振峰显著增强,即表明提取的胡敏酸为类胡敏酸的木质素;随着腐解的进行,胡敏酸的红外光谱的吸收峰强度皆显著增强,核磁共振光谱的烷基、芳基和羰基的共振峰增强,即表明此时的胡敏酸已是以木质素残体为核心并结合烷基、酰胺以及糖类物质反应形成的高分子聚合体;稻草腐解的后期,胡敏酸的红外光谱的2933cm^-1处的吸收峰强度减弱,1651、1599、1508、1461、1422和1224cm^-1处的吸收峰小幅增强,核磁共振光谱的烷基共振峰减弱,甲氧基共振峰增强,表明此时的胡敏酸发生脱烷基（主要是甲基）过程。因此,红外光谱吸收峰强度与核磁共振光谱共振峰强度的规律性变化反映了稻草腐解过程紫色水稻土胡敏酸的形成过程具有阶段性,紫色水稻±胡敏酸的形成过程符合木质素学说。     

The sodium-amalgam reduction of soil and fungal humic substances

Two soil humic acids and a “humic acid” synthesized in the laboratory by Stachybotrys chartarum were reduced with Na-amalgam. The reduction products were methylated, separated by preparative gas chromatography and identified by matching their mass and micro-infrared spectra with those of authentic specimsens.Yields of reduction products identified ranged from 2.7 to 4.2% of the initial weights of the humic materials. Major products identified were N-methyl-benzylsulfonamide, methylated phenolic acids, aromatic aldehydes and C₆C₂ - and C₆C₃ - compounds with 0 in the side chains. Since the Na-amalgam reduction of both soil and fungal humic materials produces the same or similar compounds, the method provides little information on the origin of these compounds, that is, whether they are lignin- or flavonoid-derived or synthesized by microorganisms. Compared with oxidative degradation methods, Na-amalgam reduction appears to be inefficient and tells little about the chemical structure of humic acid polymers.     

秸秆生物反应堆与菌肥对温室番茄土壤微环境的影响

为研究秸秆生物反应堆、微生物菌肥及两者配套措施对土壤理化性质和微生物功能多样性,以及作物生长的长期影响,试验以传统种植方式为对照(CK,常规栽培),研究了菌肥(T1,微生物菌肥4 kg/667 m2)、内置式秸秆生物反应堆(T2,秸秆(4 t/667 m2)+发酵沟菌剂(8 kg/667 m2)+腐熟猪粪(600 kg/667 m2))及2种措施配套处理(T3,秸秆(4 t/667 m2)+发酵沟菌剂(8 kg/667 m2)+微生物菌肥(4 kg/667 m2)+腐熟猪粪(600 kg/667 m2))对土壤理化性质和微生物功能多样性的影响。结果表明:1)与CK相比,秸秆生物反应堆能够在一定时期内提高土壤含水率;而菌肥能够在一定时期内降低土壤含水率,秸秆生物反应堆能够显著降低土壤酸性和电导率(EC,electrical conductivity)值,缓冲土壤酸化和次生盐渍化;而单施菌肥对土壤酸碱性和EC值没有显著影响。2)秸秆生物反应堆(T2)增加了土壤中有机质的含量和土壤微生物量,降低土壤中速效磷、钾的含量;微生物菌肥(T1)降低了土壤中有机质含量和微生物量,而显著提升了土壤的速效磷、速效钾含量,两种措施配套处理效果则更明显。3)菌肥能够改善土壤微生物对多聚物、碳水化合物和氨基酸的利用效率,而秸秆生物反应堆能够促进土壤微生物对于一部分氨基酸、羧酸类、酚酸类和胺类物质的利用。而2种措施同时使用时,其促进和改善微生物碳代谢能力的作用则更加显著。4)各处理均能够在一定程度上增加各年度番茄产量。综合考虑,认为内置式秸秆生物反应堆和菌肥配套处理(T3)能够更好的改善和修复日光温室连作土壤,增加作物产量,是一种较为有效的农艺措施。     

微生物关键种影响植物残体还田条件下木质素酚浓度——基于30年长期有机培肥试验

探究不同植物残体还田条件下微生物关键种如何影响木质素酚的浓度(木质素酚与土壤有机碳的比值),旨在为不同有机物料还田培肥土壤提供理论和技术依据。本研究以河西绿洲灌区的灌漠土作为研究对象,开展了30 a以施加绿肥(GM)、小麦秸秆(SW)、氮肥(N)、绿肥配施氮肥(GMN)、小麦秸秆配施氮肥(SWN)以及不施肥(CK) 6种不同施肥处理的培肥试验。结果表明:GM和SW处理的土壤有机碳含量显著高于其他处理;有小麦秸秆参与还田的处理显著提高了总木质素酚浓度以及3种木质素酚类单体的浓度,其中总木质素酚浓度在SW处理中分别比CK、GM、N、GMN和SWN处理高了109%、122%、115%、164%和57%;对比不施肥和施化肥处理,植物残体还田显著提高了微生物关键种F3(盘菌纲,Pezizomycetes)、F26(被孢霉菌纲,Mortierellomycetes)、F173(粪壳菌纲,Sordariomycetes)、B74(阿尔法变形菌纲,Alphaproteobacteria)和B87(放线菌纲,Actinobacteria)的相对丰度。本研究共鉴定出15个关键种,其中关键种B831(属阿尔...     

Stability and mineralization of organic phosphorus incorporated into model humic polymers

The stability of the phosphate ester linkage in phosphoserine (PS) and phosphoethanolamine (PE) was evaluated after incorporation of these compounds into model humic polymers. Humic polymers prepared by oxidation of a mixture of substituted phenols in the presence of either PS or PE resulted in model humic materials containing from 0.25 to 0.94% P, values within the range found for organic P in natural soil humic materials. The organic P contained in model humic polymers was resistant to hydrolysis with 1 n HC1 and 1 n NaOH and resistance of the P ester to hydrolysis with 6 n HCl was increased through incorporation into model humic polymers. Organic P in model humic polymers was also stabilized towards hydrolysis with acid and alkaline phosphomonoesterases. Less than 11% of the organic P in polymers containing PS and PE was hydrolyzed by acid or alkaline phosphatase. The incorporation of PE into a model humic polymer markedly reduced the amount of P mineralized during incubation in soil when compared to P mineralized in soils treated with PE. For all environmental conditions imposed during soil incubations (i.e. pH, aeration, temperature), only 20% of the P in model humic polymer containing PE was released during a 16-week period. In contrast, > 60% of the P in either PS and PE added individually to soils or PS and PE intimately mixed with preformed model humic polymer and then added to soils was released during the initial 7 days of soil incubation. The results suggest that a portion of the unidentified organic P in soils may arise from the incorporation of organic compounds containing both amine and phosphate ester functional groups into humic materials and that the organic P thus formed is resistant to both chemical and enzymatic hydrolysis.     

The contribution of lignin to the composition of humic acids from a wheat-straw amended soil during 3 years of incubation in pots

An Andisol from the Vesuvian volcanic area, low in organic matter, was treated with wheat straw (20/1 w/w) and urea (500/1 w/w) in pots, and maintained at 60–90% of field capacity in an open greenhouse in order to study the yields and properties of humic acids after 30, 122 and 224 d, and 1, 2 and 3 years of incubation. During the first year the yields increased, but subsequently they showed a decrease. However, the chemical and spectroscopic properties of humic acids remained substantially constant. The chemical characteristics of modified lignin from added wheat straw were recognized in the properties of humic acids by UV-visible, FT-IR and ¹³C CP-MAS NMR spectroscopic analysis. Evidence was obtained that these modifications included the microbial removal of carbohydrates, the oxidative destruction or transformation of the side chains of lignin units, the demethylation of mainly guaiacyl, and subsequent oxidation of catechol-like structures to o-quinone. The latter can be postulated to promote the nucleophilic addition of amino compounds with formation of aromatic amines in humic acids.     

The problem of water hyacinths in rivers and canals

Abstract

The cation exchange capacity (CEC) and content of COOH groups in bulky water hyacinth compost were estimated over the rotting period. The progressive increase observed in the estimates was ascribed to continuing oxidation processes in the composted materials.

Fractionation of organic carbon extracted with Na₄P₂O₇-NaOH from the composted materials over the rotting period on the basis of acid-soluble and -insoluble fractions, was carried out. The carbon of humic-like substances (precipitated at pH 2) in the dry matter was almost stable from 0- to 124-day composted materials. There were successive increases in total acidity, and content of carboxyl and phenolic hydroxyl groups in the humic substance extracts indicating progressive oxidation processes in these substances over the rotting period. Elementary analysis and IR spectra provided fürther support for the above inference and led us to conclude that humification of the water hyacinth consists mainly of structural modification of humic-like substances initially occurring rather than a condensation of simple organic carbon to form high molecular weight material. Such structural modification is dominated by biochemical oxidation and increases in oxygen-containing functional groups. This conclusion is consistent with the view that “humic acids are formed by processes involving oxidation of plant lignin and lignoprotein entering the soil.”     

Decomposition of 14C-labeled lignins,model humic acid polymers,and fungal melanins in allophanic soils

During 1 yr, CO₂-C losses from 7 agricultural soils containing 0.5–1.5% organic C ranged from 70 to 243 mg 100 g ^{? 1} while losses from three allophanic soils containing 4.9–8.9% organic C varied from 92 to 191 mg. Losses as ¹⁴CO₂ from ring-labeled model and cornstalk lignins averaged about 30% from the agricultural soils compared to about 11% for the allophanic soils. Losses of 2-side chain lignin carbons were about the same as for the ring carbons. Carbon losses from 1-side chain and methoxyl C varied from 42 to 59% in the normal soils while losses from the allophanic soils were a third to a half these values. From 6 to 9% of protein, cysteine, lysine, and glucosamine carbons linked into model humic acid polymers were lost during 1 yr in the allophanic soils compared with 13–24% from the normal soils. Comparable losses from two fungal melanins were 7–15% for the normal soils and 2–4% for the allophanic soils.     

Colluvisols under cultivation in Schleswig-Holstein. 3. Soil organic matter transformation after translocation

‘Colluvisols’ (Colluvi-cumulic Anthrosols) are an important soil unit in North Germany. In the landscape of loamy till these soils are associated with eroded Luvisols. The soil organic matter (SOM) of top layers of both soils was compared by using approaches of wet chemistry, CPMAS ¹³C-NMR and pyrolysis field-ionization mass spectrometry (Py-FIMS). The Luvisols are sources of SOM transfer due to a continuous erosion process. The annual input of straw and plant residues induces the dominance of litter compounds like proteins, polysaccharides and lignin in the SOM. The Colluvisols are sinks of SOM transfer with a predominance of humic compounds. Lignin is degraded forming humic compounds with an alkylic and aromatic structure. In these soils selected compounds with higher mass signals were detected by Py-FIMS, which may indicate the existence of typical “SOM markers” in the colluvic materials.     

Influence of clay minerals on the formation of humic substances by Epicoccum nigrum and Stachybotrys chartarum

The influence of montmorillonite, kaolinite and finely ground quartz on the formation of humic acid-type polymers by Epicoccum nigrum and Stachybotrys chartarum was studied. The fungi were grown in shake and in deep (4 cm) or shallow (1.5 cm) stationary cultures. In general, clay shortened the time required for the formation of dark colored substances and increased the amounts of humic acid-type polymers in the culture solutions or extracted from the cells with NaOH. In some tests, the time of maximum humic polymer accumulation was much earlier in the presence of clay but total amounts formed in check cultures eventually equalled that of the cultures with clay additions. The ratios of the total humic acid to the total cell substance or to the glucose consumed were also generally increased by montmorillonite. Kaolinite and quartz exerted a similar but less pronounced effect. In deep stationary cultures of S. chartarum, total growth and humic acid formation was sometimes depressed by the higher concentrations of montmorillonite but in shallow cultures biomass and humic acid formation were increased. In cultures with an initial pH of 6.0, humic acid polymers were formed in the cells before they appeared in the solutions. During autolysis, some of the cellular polymers were either released into the medium or became more readily extractable with NaOH. The clays did not appreciably alter the chemical properties of the humic polymers, namely, C and N contents, exchange capacity, COOH groups, total acidity, or phenols released upon sodium amalgam reduction. In the presence of clays, phenols were formed more quickly in the culture solutions, but the kinds and relative amounts did not appear to be altered. Clays did not significantly affect oxygen consumption during autoxidation of phenol mixtures. The observations indicate that, by affecting growth and metabolism, the clays indirectly influence phenolic polymer formation.