Partial Characterization of Soil Humic Acids Through Biodegradation1

PARTIAL CHARACTERIZATION OF SOIL HUMIC ACIDS THROUGH BIODEGRADATION1

S. P. A~IATHUR~AND E. A. PAUL Deparln~enlof Soil Science, University of Saskatchewan, Saskaloon, Saskatchezoan Received October 12, 1966

A strain of Pewicillizi?tz freqzientans was successfully employed for partial degradation and characterization of humic acids. Salicyl alcohol and salicylalclehyde were detected in culture filtrates of the fungus ~rtilizinghumates under reduced oxygen tension. The enzyme systems iiivolved in the degradation of humic acids were adaptive. The humate-adapted lnyceliirln was capable of metabolizi~~ga number of co~npoundswhich occur in soil as products of degradation of li~nir~,aromatic amino acids, and plant glycosides but not polyphenolic hydrocarbons, resorcinol, and phlorogluci~~ol.

Introduction In a previous paper the authors suggested the use of biodegradation for determining the structural features of hurnic acids and reported isolation of a number of organisms capable of utilizing these acids (7). One such isolate, a strain of Penicilliunz frequentans, degraded 32% of huinic acids in replace- ment-shake culture (7). The organisin appeared to use the predominantly aromatic 'core' of humic acids as a source of carbon (7). This paper reports an attempt to characterize a portion of the humic acids through identification of the initial products of their degradation by the penicillium. Materials and Methods Hzimic Acids The 'mobile' humic acids used in these experiments were extracted from the Ap horizon of a Allelfort Orthic Black soil by standard alkali and acid fraction- For personal use only. ation technique (1). These huinic acids were found, by 14C dating, to have a mean residence time of 785 + 50 years (1). Ether Extractions Control and incubated cultures were extracted with diethyl ether at pH 2.0, 7.0, and 9.0 (obtained by using 3 N HCI and 3 N NaOH) for 24 hours at each pH. The ether extract was evaporated in vaczlo and remaining traces of ether driven off with nitrogen gas. Anhydrous sodium sulfate was added to absorb remaining traces of moisture and to render the small amounts of humic acid present insoluble in the subsequent extractant, anhydrous diethyl ether. Paper Chromatography One-way descending paper chromatography of the ether extracts was carried out on Whatman No. 1 filter paper using benzene: acetic acid :water (100 :25 :25 v/v), or formic acid saturated benzene (12, 9). Diazotized sulfanilic acid (12), p-nitraniline (12), ferric chloride (9), and 2,4-dinitrophenyl hydrazine were used as detection reagents (9). 'Saskatchewan Institute of Pedology Publication R 10. 2Present address: Soil Research Institute, Canada Department of Agriculture, Ottawa, Ontario. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14 Canadian Journal of hlicrobiology. Volume 13 (1967) 582 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 13, 1967 Gas Chromatographic Analysis The culture extract was injected into the chromatograp11 (Aerograpll-90 Model) fitted with a 6-ft long copper column packed with 10% (\v/w) Apiezon M grease on fluoropak SO. Helium was used as the carrier gas at a flow rate of 75 ml/min. The temperatures of the sampler, col~~mn,and detector were maintained at 230 "C, 210 "C, and 240 "C respectively. Adabtation of the Penicilli~~mto fJ~lmic Acids Respironletric studies were undertalien to determine if the penicillium could be adapted to the utilization of humic acids. Sterilized Czapel; Dox broth \\as inoculated \vitli spores of the penicillium and shalten for 72 liours at room temneraturc. i\Ivcclium harvested from this \\-as used for rcsniro- metric studies either directly (as non-adapted mycelium) or after being in contact wit11 Iiumic acids as sole source of carbon for 18 liours (adapted

mvcelium). Humic acids \\;ere sunnlied& & as sole source of carbon to the two sets of myceliu~i~in Warburg flasks and oxygen consumption measured. The basal medium \\-as sugar-free Czapck Dox broth and total volume of cultures in the flasks was 3.0 ml. Results \TThen introduced for the first time as a source of carbon, humic acids initially suppressed the respiration of nlycclium (Fig. 1). This inhibition For personal use only.

1234567 TIME (hours) Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14 FIG. 1. Oxidatioll of humic acids by h~uuate-adapted(-) and non-adapted (---) myceliu~u(endoge~lous substracted). MATHUR AND PAUL: SOII, IIUMIC ACIDS 583

lasted about 5 hours and \\,as follo\ved by a gradual adaptation to the substrate. The pre-induced ~nyceli~~nloxidized the humic acids directly. These results indicated that an adaptive enzyme systelll was employed by the penicillium in the degradation of humic acids. Further studies were conducted to determine if the humate-induced penicillium was also adapted to a number of aroinatic com~ounds.Thcse corn- pounds have been reported to IIC components of l~umusor are kno\vn to be kej7 intermediates in the degradative pathways of such substances (14, 6, 3, 2, 15). ,411 tested substrates provided 0.1% carl~onin the medium. During adaptation to humic acids the fungus also acquired the aljility to metaljolize catechol, salicylate, tyrosine, protocatechuate, guaiacol, vanillin, phenylpyru- vate, and ferulic and syringic acids (Fig. 2). Humate-induced mycelium failed to metabolize the following: a-conidendrin, rutin, nz-OH benzoate, pliloretic acid, pyrogallol, naphthalene, phenanthrenc, anthrace~le, pl~loroglucinol, anthranilatc, tryptophai~,p-crcsol, cin~~amicacid, indole, resorcinol, niacin, coun~arin,wood pulp, cellulose, phthalic acicl. Chromatographic studies of ether estracts of the pcnicillium cultures containing 0.2% humic acids as the sole source of carbon periodically gave rise to one spot detectable with diazotizcd sulfanilic acid (light yellon, to golde~z), p-nitraniline (mauve), and ferric chloride (light violet). These results were inconclusive hecause of the presence of large amounts of unresolved material. Ho\\~ver,salicyl alcohol and salicylalclehyde were easily resolved on chromatography of the extracts of culture filtrates of 110th adapted and non-adapted mycelia growing on humic acids for S to 24 hours at reduced oxygen tension (10% of atmospheric). Identificatio~~was confir~medby gas chromatography where evtracted material gave rise to the same peal; as the l;no\\~ncompounds (Fig. 3). These compounds were not detected, by paper or gas cllron~atograplly, For personal use only.

1234567 1234567 TIME (hours) TIME (hours) Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14 FIG. 2. Oxidation of various substrates by humate-adapted (-) and non-adapted (---) rnyceli~rln(cadogeno~~s substractcd). CAN;\Dl.\N JOURNAL OF MICROBIOLOGY. VOL. 13, 196i

STANDARDS CULTURE EXTRACTS

dl23456789 'dl 2345678B TIME (mid TIME (mid FIG. 3. Identification of intermediates by gas chromatography.

in the ether extracts of humic acids, the inycelium, or in filtrates of sucrose- based cultures of the penicillium. It was estimated that salicyl alcohol and salicylaldehyde accounted for about 575 of the carbon of hulnic acids under- going degradation under the reduced oxygen tension. Similar experiments were carried out under conditions of reduced oxygen tension (50% of atmospheric) to determine if the humate-induced mycelium was adapted to the use of salicyl alcohol. In these studies the pre-induced mycelium was found capable of inetabolizing salicyl alcohol giving rise to products identified as salicylaldehyde, salicylic acid, and catechol (Table I). Salicyl alcohol was not metabolized by the non-adapted mycelium in the 1- to

For personal use only. 4-hour incubation periods. Results of paper chromatographic analyses were checked against standards and also found to be in agreement with known values (9). The possibility that salicyl alcohol was derived from salicylic acid rather than directly froin the humic acids was also investigated. Salicylic acid was supplied as the sole source of carbon to the mycelium adapted to humic acids. under conditions of both normal and 10% normal oxygen tension. No salicyl alcohol was detected in the ether extract of these cultures. TABLE I Identification of aromatic products of degradation of salicyl alcohol by the humate-adapted penicillium (paper chromatography, using benzene:formic acid solvent*)

Observed Con~pounds R, values Color with FeC13 Catechol 11 Green to black Salicyl alcohol 2 2 Light violet Salicylic acid 55 Dark violet Salicylaldehyde 67 Light red Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14 'Rtio (9). hl.4TIIUR .4XD P.lUL: SOIL IIUMIC ACIDS 585 Discussion A previous study showed that Penicillizim freqzientans, isolated from soil, was capable of partially degrading the aromatic portion of soil humic acids (7). The experiments reported here denlonstrated that this degradation was brought about by an adaptive enzyme system. Such a systein offered a pro- cedure for determining whether any of the several kinds of compoundi re- garded as possible components of huinic acids could have been part of the portion utilized by the penicilliuin. Siinultaneous adaptation studies revealed that the humate-induced iny- celiuin did not possess the ability to degrade polyphenolic hydrocarbons, resorcinol, and phloroglucinol. It may, therefore, be assumed that the portion of humic acids utilized by the fungus did not consist of polycyclic hydrocarbons or the trihydric phenols. During growth on hurnic acids the fungus did become adapted to a nuinber of coinpounds which occur in soil as products of degradation of lignin, aromatic ainino acids, or plant glycosides. It is probable that these substances, their precursors, or their derivatives con- stituted that part of the humic acids which was utilized by the penicilliuin. Direct evidence of the nature of the degraded portion of huinic acids was provided by the detection of salicylaldehyde and salicyl alcohol in cultures of the penicillium utilizing humic acids as the sole source of carbon. The aldehyde was probably not directly derived froin the humic acids but occurred as a ~roductof salicvl alcohol. Salicyl alcohol and salicylaldehyde may occur in soil as transformation products of aromatic compounds such as the plant glycosides salicin and populin (8). Two of the properties of salicyl alcohol and salicylaldehyde strongly suggest a role for these compounds, and their hoinologues, in humus synthesis. They undergo self-condensation to give compounds containing For personal use only. ether linkages and forill substitution products with ainino coinpounds (13, 10, 5). These studies demonstrate the applicability of biodegradation in the characterization of soil humus. Since enzymes are specific and their reaction conditions mild, products of their activity should reflect the structure of humic acids more faithfully than the altered products of chemical degradation. Salicylaldehyde and salicyl alcohol are the first coinponents to be recog- nized through the controlled biological degradation of humic acids. Both substances are cheinically active and would be readily oxidized to salicylic acid or its derivatives during oxidative chemical degradation of humic acids. Salicylic acid and its derivatives, e.g. picric acid, have been previously found in the oxidation products of soil humus (4, 11). High temperatures, necessary for hydrogenolytic depolymerization, and alltali treatments would result in conversion of salicyl alcohol and its aldehyde into resinous substances which cannot be identified easily (3, 5). Acknowledgment The financial assistance (EMR Grant No. 130) provided by the Canada Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14 Department of Agriculture is gratefully acknowledged. 586 CANADIAN JOURNAL OF MICROBIOLOGY. VOL. 13, 1967 References 1. CAMPBELL,C. 11. 1965. The use of naturally occurring Cl1 to nieasure the persistence of organic co~nponentsin soil. PI1.D. Thesis, Dcpt. Soil Sci., Univ. of Saslratchewan. 2. EVANS,W. C. 1963. The microbiological degradation of aronlatic co~ilpounds.J. Gen. Microbial. 32, 177-184. 3. FELBECIC,G. 'T., JR. 1965. Structural chemistry of soil hurnic substances. Advan. rlgron. a- ",.* -,- I!, 5Ll-508. 4. HAY.~SAI,T. and NAG.\I, T. 1960. On the components of soil humic acids. VIII. The oxidative decomposition by all~aline potassi~~n~per~nanganate and nitric acid. Soil Plant Food Tolcyo. 6, 170-175. 5. I

SPRUXG.ibI. hI. ant1~ GLADSTOVE.~ I<. 1949. A st-II~V... .~of solnr contlensations of o-nlethylo1- M. , - --~~--- phhnols. J. ilni. CI~~L.~SO~.71, 2907-2913. S~EELIXI~,C. 1963. \\:hat is h~lmicacitl? J. Chc~n.E~IIC. 40, 379-384. Towens, G. 1-1. N. 1964. i\~Ietabolismof phenolic compounds. 117. Biochemistry of phenolic compounds. Edited by J. B. I-Iarborne. ilcademic Press, Se\\l York. pp. 272-294. For personal use only. Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by COLORADO STATE UNIV LIBRARIES on 01/15/14