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

Soil Biol. Biochem. Vol. 4, pp. 147-154. Pergamon Press 1972. Printed in Great Britain

INFLUENCE OF CLAY MINERALS ON THE FORMATION OF H U M I C SUBSTANCES BY EPICOCCUM N I G R U M AND S T A C H Y B O T R YS C H A R T A R U M Z. F1LIP* a n d K. HA[DER Institut fiir Biochemie des Bodens der Forschungsanstalt fiJr Landwirtschaft, Braunschweig, Germany a n d J. P. MARTIN Department of Soil Science and Agricultural Engineering, University of California, Riverside, California

(Accepted 7 July 1971) Summary--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. INTRODUCTION THE AUTHORS have shown (Filip et al., 1972) that the a d d i t i o n of small a m o u n t s of clay, especially m o n t m o r i l l o n i t e , to well-aerated cultures of Epicoccum nigrum a n d Stachybotrys chartarum, fungi which synthesize h u m i c acid-type polymers, greatly accelerated cell synthesis a n d t r a n s f o r m a t i o n of nutrients. The m e c h a n i s m of the h u m i c acid f o r m a t i o n ( M a r t i n a n d Haider, 1971) involves the synthesis of orsellinic acid a n d p - h y d r o x y c i n n a m i c acid-type phenols which are altered t h r o u g h i n t r o d u c t i o n of a d d i t i o n a l methyl a n d hydroxyl groups to the ring, oxidation of side chains, oxidation of methyl groups, or decarboxylation to f o r m n u m e r o u s phenols. M a n y of these u n d e r g o oxidative polymerization to f o r m the h u m i c acid-type polymers. U n d e r n a t u r a l conditions, higher quantities of h u m u s are often associated with soils c o n t a i n i n g relatively larger quantities of clay. This p h e n o m e n o n also occurs in mixed * Present address: Department of Microbiology, University of Agriculture, Prague 6-Suchdol, Czechoslovakia. 147

148

Z. FILIP, K. HAIDER AND J. P. MARTIN

culture studies involving sand-clay mixtures. Ziemiecka and Kobus (1960) observed that the addition of bentonite to sand-plant residue mixtures reduced nitrogen losses and increased the humus content. Filip (1968, 1969) studied the influence of bentonite on the formation of humic substances by a mixed soil flora in sand and soil cultures. Casein was used as the C and N source. The clay greatly increased the humus content of the cultures. In mixed culture studies, involving sand-clay mixtures or natural soils, it is not known whether an increase in humic acid noted after a prolonged incubation period is associated with an effect of clay on growth and metabolic activity of the organisms involved in the humification process or whether a strong adsorption of humic substances by the clay which reduces the decomposition rate of the humus is the main factor involved. The purpose of this study was to determine the influence of clays on the processes involved in the formation of humic substances and on the amounts of humic polymers synthesized by E. nigrum and S. chartarum.

METHODS

Details of the cultivation procedures and the source and nature of the clays or colloids used were reported by Filip et al. (1972). The methods for recovery and purification of the humic acids in the culture solutions have been described (Martin et al., 1967). Briefly, this involved filtration or centrifugation, and dialysis of the culture solution. The humic acid was precipitated by adjusting to pH 2, recovered by centrifugation, washed with 0.01N HC1 and dried by lyophilization. This fraction is designated as HA 1. A second fraction (HA 2) was recovered from the cells of the cell-clay mixtures by extraction with 0.5rq N a O H and precipitation at pH 2. The C content of the soluble fraction is referred to as FA 2. Before acidification, the clay was flocculated by the addition of N a z S O 4 and removed by centrifugation. Usually, a greater amount of humic acid-type polymer was recovered by 0-5N N a O H extraction if the cell material was first hydrolyzed for 2 hr at 90°C with 1N H z S O 4. The total HA isolated by this procedure minus HA 2 was designated HA 3. Portions of the HA preparations were placed in a combustion furnace at 600°C, and the ash contents subtracted to obtain the HA weights. For analysis of inorganic constituents, the preparations were digested in 30 % (v/v)H202. Exchange capacity, carboxyl groups, and total acidity were determined as described by Martin et al. (1967) and Schnitzer and Gupta (1965) and C was determined by a wet combustion method with chromic acid (Allison et al., 1965). The spectra of the humic acids were measured over the range of 350 to 675 nm. The values at 475 and 650 nm were selected for calculation of the color quotient, Q,/6. Reductive degradation of the polymers with sodium amalgam and separation of the phenols released, using thin layer chromatography, were carried out by the methods of Burges et al. (1964) and Martin et al. (1967). Phenols formed in the solution cultures were extracted and identified, and O2-uptake of phenol mixtures in the presence of montmorillonite were determined as described by Haider and Martin (1967). RESULTS After 15-20 days of incubation, both shake and stationary cultures ofE. nigrum gradually turned red-brown and then dark brown as the humic acid-type polymers formed. The addition of montmorillonite accelerated the color change, and the intensity of the dark color was proportional to the concentration of the clay (Fig. 1). For these measurements, the solutions were centrifuged at 15,000 g, appropriately diluted and recalculated for 1 cm

INFLUENCE OF CLAYS ON FORMATION OF HUMIC SUBSTANCES BY SOIL FUNGI

149

o p t i c a l p a t h w a y . T h e s t a t i o n a r y culture solutions were m u c h d a r k e r a n d c o n t a i n e d m u c h higher a m o u n t s o f h u m i c acid which c o u l d be recovered by p r e c i p i t a t i o n at p H 2. T h e a c t u a l a m o u n t s o f h u m i c acid recovered f r o m the m e d i u m a n d the cells after 20 a n d 30 days o f i n c u b a t i o n for the s t a t i o n a r y cultures are i n d i c a t e d in T a b l e 1. D u r i n g the earlier p e r i o d s o f i n c u b a t i o n , the a m o u n t s o f humic acids f o r m e d were increased in a m a n n e r similar to increases in b i o m a s s (Filip et al., 1972).. T h e synthesis o f the h u m i c - t y p e p o l y m e r s c o m m e n c e d earlier in the m y c e l i u m o f this culture t h a n in the culture m e d i u m . Also, the H A 3 fraction decreased with time with increasing m o n t m o r i l l o n i t e , while solution h u m i c acid ( H A 1) increased. The F A 2 fraction also decreased, but, in a d d i t i o n to possible humic acid precursors, this fraction c o u l d c o n t a i n m a n y other substances. T h e a m o u n t s o f humic acids isolated f r o m the cells o f the shake cultures showed a similar p a t t e r n to those o f the stat i o n a r y cultures, b u t the a m o u n t s recovered f r o m the solution were greatly reduced.

o • tx •

/

control 025 % clay 05 % clay I'0 % clay

/

2'5 o :

2.0.

contro[ 0.25 % clay 05 % Clay 1,0 % c[~y

/• /

4

/

/e A

~"

/

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1.5.

.J

I0.

J

o~ .=

,~ 0"5.

(b)

Ca)

lo

Days

zb

ib

3'o

go

3b

Days

Fxo. I. (a) Absorption at 475 nm of the shake and (b) deep stationary culture solutions of E. nigrum at various montmorillonite concentrations.

TABLE 1. FORMATION OF HUMIC SUBSTANCES IN STATIONARY CULTURES OF E. nigrum WITH DIFFERENT CONCENTRATIONS OF MONTMORILLONITE FROM BED ROCK, WYOMING

20 days Montmorillonite ( ~ w/v) Humic acid From culture solution (HA 1) From cells extracted with NaOH (HA 2) From cells after acid hydrolysis (HA 3) Total 'Fulvic acid'

0

0

0"25

0"5

30 days 1

0

0"25

0.5

1

Grams of ash-free and dry humic acid in 1 1. of culture medium 0 0"24 0"88 0.12 0"38 0"57 0-98

0

0"66

0 0

0" 54 1"20

0"87

0"80

0-32

0"10

0"73

0"78

0"47

0- 45 0" 02 0 0" 15 1"49 1-22 0-22 1"76 g carbon/1 in supernatant of HA 2 0"70 0"49 0"33 0-95 0-48

0 1"35

0" 18 1"63

0"44

0"23

150

Z. FILIP, K. HAIDER AND J. P. MARTIN

In order to show more clearly the extent to which the energy source, glucose, was converted to humic acid-type substances relative to cellular substance, the ratios of total humic acid formed to glucose consumed and the ratios of total humic acid to total cell substance synthesized were calculated (Table 2). After 20 and 30 days incubation, the presence of montmorillonite greatly increased these ratios.

TABLE 2. RATIO OF HAToT" X 100 (TABLE 1) TO BIOMASS OF E. nigrum IN STATIONARY CULTURES AND TO GLUCOSE CONSUMED

20 days Montmorillonite additions (%w/v) Control 0"25 0"5 1"0

30 days

g of HAtot. x 100 per lg cells per lg glucose 0 18"2 15"7 15.8

g of HAtot. x 100 per lg cells per lg glucose

0 4"7 5-8 4"1

4-1 17"5 18-0 29.6

1"5 4"2 4"5 5"4

Kaolinite and ground quartz also increased or accelerated humic acid production, but, in general the magnitude of the effect was only about half to two thirds that obtained with montmorillonite. In these tests, the humic substances were also formed in the cells before they were noted in the medium. During the later periods of incubation, the humic polymers tended to decrease in the cells and increase in the medium. Maximum humic acid formation occurred at the 0.5 per cent mineral addition. In general, the ratios of total humic acid formed to total cell weight or glucose consumed were higher in cells from cultures containing clay. S. chartarum synthesized much larger quantities of humic acid-type polymers than E. nigrum (Table 3) and, in contrast to E. nigrum greater quantities were found in the shake culture solutions than in the deep stationary cultures. In both types of culture, much higher quantities were formed in the cells. With S. chartarurn much higher quantities ot humic acid were formed in the shallow stationary culture solutions than in the deep stationary or shake cultures (Table 4). The addition of montmorillonite greatly accelerated the formation of the polymers, but, with time, the amount found in the controls was similar to that found in the cultures containing clay.

TABLE 3. FORMATION OF HUMIC SUBSTANCES IN SHAKE CULTURES OF S . chartarum WITH DIFFERENT CONCENTRATIONS OF MONTMORILLONITE (MOOSBURG~ GERMANY) AFTER 15 DAYS INCUBATION

Montmorillonite (% w/v) Humic acid From culture solution (HA 1) From cells extracted with NaOH (HA 2) From cells after acid hydrolysis (HA 3) Total

0

0" 25

0" 5

1

Grams of ash-free and dry humic acid in 1 1. of culture medium 0.49 0.86 1-31 1.62 3"61 4"00 4"60 4"55 0" 68 0" 55 0" 36 0"07 4" 78 5"41 6"27 6" 24

INFLUENCE OF CLAYS ON FORMATION OF HUMIC SUBSTANCES BY SOIL FUNGI 151 TABLE 4. INFLUENCE OF MONTMORILLONITE (UPTON, WYOMING) ON ACCUMULATION OF HUMIC ACID IN SHALLOW GLUCOSE-ASPARAGINE CULTURE MEDIUM OF S. chartarum

Montmorillonite addition (% w/v)

Days

10

20

30

0-2 0-5 0.6 0.7

1.1 2.8 2.7 2-5

50

Humicacid(g/1) 0 0.25 0.50 1"~

2.2 3"4 3.0 2.6

3.2 3.4 2.9 2.0

During the early stages of growth of both E. nigrum and S. chartarum, the quantities of humic acid which could only be extracted from the cells following acid hydrolysis were relatively high but decreased with time. The quantities which could be extracted with 0.5N N a O H without a hydrolysis pretreatment, however, increased as did the amounts in the solution culture. The presence of clay accelerated these changes. The ratios of total humic acid to glucose consumed and total biomass for shake cultures of S. chartarum are presented in Table 5. These ratios increased with increasing clay additions. Similar calculations for deep stationary cultures containing 0.5 and 1.0 per cent (w/v) montmoriUonite from Upton, Wyoming only slightly increased these ratios, which was expected from the less pronounced growth effects under the cultural conditions (Filip et al., 1972). TABLE 5. RATIO OF HATOT. X 100 TO BIOMASS OF S. chartarum OR GLUCOSE CONSUMED IN SHAKE CULTURES

MontmoriUonite additions (% w/v) 0 0"25 0"5 1.0

g of HAtot. X 100 p e r 1 g cells per 1 g glucose

44"7 51 "0 62"7 73"4

16"1 18"9 21"3 21" 1

The addition of quartz or kaolinite to the cultures caused an intermediate increase in humic acid formation, especially of humic acids that were extracted from the cells. The ash contents of the humic acids from E. nigrum cultures containing montmorillonite were very high and ranged up to 60 per cent for the 1 per cent clay treatments, even though the solutions were first centrifuged at 15,000 g before precipitation of the polymers. The S. chartarum humic acids obtained by the same procedures, however, contained only 3-4 per cent ash when clay was present in the cultures. When the culture solutions of both fungi were altered to remove clay and cells, the ash contents ranged from 4 to 20 per cent. Infrared analysis of the high ash humic acids indicated that the ash was primarily clay. The E. nigrum humic acids consistently contained about 54 per cent C and 8 per cent N on an ash-free basis. The addition of clay to the cultures did not affect these percentages. Also, the clay exerted no effect on the exchange capacity, the COOH groups or total acidity (see Martin et al., 1967). The clays also did not affect the properties of the S. chartarum humic acids. These contained about 56 per cent C and 5-6 per cent N. s.B.s. 4 / 2 - - c

152

Z. FILIP, K. HAIDER AND J. P. MARTIN

The spectra of the humic acid preparations were measured in the 675 nm range. The humic acids from the cells and solution cultures of E. nigrum showed an increase in the slope of the curve with increasing montmorillonite additions to the culture solutions. The S. chartarum polymers were not affected by the clay, but the slope tended to increase with increasing age of the culture. The latter spectra were more similar to those for soil humic acid and resembled those of humic acid extracted from Chernozem soils (Flaig et al., 1955; Kononova, 1966). Before humic acids form in the culture solutions of these fungi, various phenols appear (Haider and Martin, 1967; Martin and Haider, 1969). The presence and relative quantities of the various phenols were estimated by the size and intensity of the spots on two dimensional thin-layer chromatograms from ether extracts of acidified culture solutions. In general, the clay additions did not affect the kinds or amounts of phenols synthesized, but, under conditions of accelerated growth the phenols were synthesized more quickly. In addition, there could be some adsorption of some of the phenols by the clay under acid conditions as noted by Flaig et al. (1971). Phenols released upon sodium amalgam degradation of the polymers were also separated by two-dimensional thin-layer chromatography. The addition of clay to the cultures did not appear to alter the kinds or amounts of phenols obtained. Haider and Martin (1967) have shown that at least part of the humic acid-type polymers formed by these fungi in the culture solution could result from an autoxidation of reactive phenols, such as 2,3,5-, 2,4,5- and 3,4,5-trihydroxytoluenes, at pH values of 6-8. These oxidize and combine with other phenols and amino acids and peptides synthesized by the fungi to form the large humic acid molecules. Respirometer and test tube studies with phenol mixtures supported this view. Heavy metal ions, such as Fe 2÷ or Cu 2 +, accelerated oxygen uptake and increased humic acid-type polymer formation. The clays used in the present experiments contain Fe and other heavy metals (Jasmund, 1955; Flaig et al., 1971). It might be expected, therefore, that the clay minerals in the presence of autoxidizable phenol mixtures could influence autoxidation by either furnishing or adsorbing heavy metals. The influence of the clays on the oxidation of phenol mixtures was, therefore, tested in Warburg and Gilson respirometers. For the respirometer tests, 5 t~moles of autoxidizable phenols, such as 2,3,5-trihydroxytoluene, plus 20/,moles of other phenols, such as orcinol, 3,4-dihydroxy toluene or 3,4and 3 5-dihydroxybenzoic acid with and without glycine or peptides, were used (Haider

TABLE6. INFLUENCE

OF CLAY ON 0 2 UPTAKE FROM REACTION MIXTURES OF 2,3,5-TRIHYDROXYTOLUENE AND 3,4-DIHYDROXYTOLUENE WITH AND WITHOUT CLAY (1 ~oo W/V)

Reaction constituents 2,3,5-trihydroxytoluene* + 3,4-dihydroxytoluenel + 3,4-dihydroxytolueneand montmorillonite + 3,4-dihydroxytolueneand vermiculite + 3,4-dihydroxytolueneand kaolinite * Five tLmolesof 2,3,5-trihydroxytoluene. t 20 t,moles of 3,4-dihydroxytoluene.

02 uptake, tzmoles, at pH 7.0 5"0 8"8 7"8 8"6 8-7

INFLUENCE OF CLAYSON FORMATION OF HUMIC SUBSTANCESBY SOILFUNGI 153 and Martin, 1967, •970; Martin and Haider, •969). The oxygen uptake of these mixtures in the presence and absence of 0.5-1.0 per cent montmorillonite or kaolinite was determined at pH values of 7 or 8. Typical results are indicated in Table 6. The clays exerted very little effect with the exception that 1 per cent montmorillonite slightly decreased Oz consumption. Additional tests not given in table form showed that the addition of Fe z+ or Cu E+ ions to the clay-phenol mixtures also exerted little effect.

DISCUSSION

The humic acid-type polymers formed by E. nigrum and S. chartarum are products of metabolism. They are primarily anabolic products formed through the condensation of acetate-malonate units to form resorcinol-type phenols and to a lesser extent through the shikimic acid pathway p-hydroxycinnamic acid and protocatechuic acid-type phenols are formed. Inasmuch as the clay minerals exerted a marked effect upon growth of the fungi, it would be expected that an effect on humic acid synthesis would also occur. A change in metabolic processes brought about by the clay might increase phenol synthesis and consequently humic acid formation. In these tests, the clay sometimes increased the total synthesis of humic polymers but sometimes the synthesis was only accelerated and, with time, the amounts of humic acid in the controls reached that formed earlier in the clay cultures. The tests where the ratios of the humic acid to biomass or glucose consumed were increased with increasing montmoriUonite are examples of the former action. According to BuLock (1967), phenols formed by Penicillium urticae are found in the greatest variety during the phase of stationary or decreasing cell dry weight. If clay minerals condense the different growth phases of an organism of this type, they could conceivably accelerate the formation of the phenols. In the investigation presented here, the oxygen supply appeared to influence the formation of humic substances. The stationary cultures of E. nigrum contained larger amounts of humic acid, but the shake cultures produced higher cell yields. Clay, however, increased humic acid formation in both types of culture. Under well aerated conditions, such as existed in the shake cultures, a greater percentage of the products of glycolysis could be used for cell synthesis while, in the deep stationary cultures, more of these products could be synthesized into phenols. As suggested by Filip et al. (1972) clay minerals appear to increase glycolytic activity of the fungus metabolism. Novak (1963) suggested that, in the soil, anaerobic processes furnish the low molecular weight precursors of phenol and humic acid synthesis while aerobic respiration furnishes the energy. This hypothesis may apply to the studies reported, because the clay minerals appear to enhance glycolysis by the fungi. Under specific conditions, this may lead to increased humic acid yields because more intermediate products would be available for phenol synthesis. This same reasoning may be applied to S. chartarum, where the addition of clay to deep stationary cultures sometimes decreased biomass but humic acid formation was not decreased proportionally. The kind of organism, the clay mineral, and cultural conditions were important in determining the extent of this effect. An important aspect of this study has been the observation that humic acids may be formed in the cells before they appear in the culture solutions. This is especially true in the cultures with KHzPO4 and an initial pH of 6.0. In the cultures with KzHPO4 and an initial pH of 7"2, however, humic polymers sometimes quickly appeared in the solution. In the thin layer cultures of S. chartarum for example, the humic polymers gradually increased in the solution. After 20 days of incubation in the 0.25 per cent clay treatment, 2.8 g/1

154

z. FILIP, K. HA1DER AND J. P. MARTIN

were present. This was also the time of m a x i m u m biomass a n d j u s t before all the glucose was utilized. Later, during autolysis, the a m o u n t of p o l y m e r in solution increased a n d was 3 - 4 g/1 at 30 days. It thus appears that, d u r i n g autolysis, some of the cellular polymers are excreted into the m e d i u m or become more readily extracted f r o m the cells with N a O H . The solution h u m i c acids can, therefore, originate from previous synthesis in the fungus cells as well as t h r o u g h a u t o x i d a t i o n of p h e n o l mixtures in the culture solution. The h u m i c polymers from the E. nigrum cultures c o n t a i n i n g 1 per cent m o n t m o r i l l o n i t e were very high in ash, n a m e l y 20-60 per cent. The removal of clay a n d cells either by centrifugation or by filtration u n d o u b t e d l y removed some h u m i c acid-type polymers adsorbed to the clay. This could have been significant, especially in the higher clay additions, so that the values for total h u m i c acid reported are p r o b a b l y low. Acknowledgements--Joint contribution of Institut f~r Biochemie des Bodens der Forschungsanstalt fiir Landwirtschaft, Braunschweig-V61kenrode, Germany, and Department of Soil Science and Agricultural Engineering, Universityof California, Riverside, California. The authors wish to thank Dr H. BEtJTELSVACrIER for furnishing clay samples, Drs W. FLAIGand H. BEOTELSVACIaERfor helpful suggestions and Miss E. PLEISS and J. O. ERVINfor skilled laboratory assistance. Dr Z. Fiuv thanks the Alexander v. HumboldStiftung for a personal stipend and Dr K. HAIOERthanks the Deutsche Forschungsgemeinschaft for a travel stipend. REFERENCES

ALLISONL. E., BOLLENW. B. and MooDm C. D. (1965) Total carbon. In Methods of Soil Analysis. (C. A. Black, Ed.) pp. 1346-1353. American Society of Agronomy, Madison. B~LOCK J. D. (1967) Essays in Biosynthesis and Microbial Development. John Wiley, New York. BUR6ESA., HtmsT H. M. and WALKI~ENB. (1964) The phenolic constituents of humic acids and their relation to the lignin of the plant cover. Geochim. eosmochim, dcta 28, 1547-1554. FILIl" Z. (1968) Development of microorganisms and humus substance formation in media with different content of bentonite. Pochvovedeniya (Moscow) 9, 55-61. (In Russian) FILIP Z. (1969) Characteristics of humic substances in a soil incubated with additions of bentonite. Rostlinna vyroba (Prague) 15, 377-390. (In Czech.) FILIVZ., HAII~ERK. and MARTrNJ. P. (1972) Influence of clay minerals on growth and metabolic activity of Epicoccum nigrum and Stachybotrys chartarum. Soil Biol. Biochem. 4, 135-145. FLAIGW., BEUTELSPACHERH. and FILIVZ. (1971) Einfluss von Tonrnineralen auf die Bildung von Huminstoffen dutch Epicoccum nigrum. Z. Pflanzenerniihr. Diing. u. Bodenk. To be published. FLAIGW., SCrtE~FERF. and KLAMROTHB. (1955) Zur Kenntnis der Huminsiiuren. VIII. Z. Pflanzenerniihr. Diing. u. Bodenk. 71, 33-37. HAIDERK. and MARTINJ. P. (1967) Synthesis and transformation of phenolic compounds by Epicoccum nigrum in relation to humic acid formation. Proc. Soil Sci. Soc. Amer. 31, 766-772. HAIDER K. and MARTINJ. P. (1970) Humic acid-type phenolic polymers from Aspergillus sydowi culture medium, Stachybotrys spp. cells and autoxidized phenol mixtures. Soil Biol. Biochem. 2, 145-156. JASMUNOK. (1955) Die Silikatischen Tonminerale. Verlag Chemie. Weinheim, Bergstrasse. KONONOVAM. M. (1966) Soil Organic Matter. Pergamon Press, Oxford. MARTINJ. P. and HAIDERK. (1969) Phenolic polymers of Staehybotrys atra, Stachybotrys chartarum and Epicoccum nigrum in relation to humic acid formation. Soil Sci. 107, 260-270. MARTINJ. P. and HAmER K. (1971) Microbial activity in relation to soil humus formation. Soil ScL 111, 54-63. MARTINJ. P., RICHARDSS. J. and HAIDERK. (1967) Properties and decomposition and binding action in soil of 'humic acid' synthesized by Epicoceum nigrum. Proe. Soil Sci. Soc. Amer. 31, 657-662. NovAl~B. (1963) Contribution to the theory of microbial formation of humus. For. Social. Agr. ScL (Prague) 12, 401-418. SCHNITZERM. and GUPTAU. C. (1965) Determination of acidity in soil organic matter. Soil Sei. Soc. Amer. Proe. 29, 276-277. ZIEMIECKAJ. and KoBus J. (1960) Influence of different compounds on the microbial activities in sandy soils. Transactions of the Seventh International Congress of Soil Science, Proceedings, Madison. 2~ 679-684.