biodegradability of dissolved organic matter in...

Biodegradability of Dissolved Organic Matter in Forest Throughfall,

Soil Solution, and Stream Water

Robert G. Quails* and Bruce L. Raines

ABSTRACTHigh concentrations of dissolved organic matter (DOM) were leached

into rainwater passing through the canopy and forest floor of an oak(Quercus spp.)-hickory (Carya spp.) forest in the southern Appala-chian Mountains. More than 95% of this dissolved organic C (DOC)and N (DON) was removed as water percolated through the soil profileand left the ecosystem in stream water. Our objective was to examinethe importance of decomposition in the removal of DOC and DON.Samples of DOM front throughfall, forest floor water, soil water fromA and B soil horizons, and stream water were all adjusted to a com-mon initial DOC concentration, inoculated with soil and stream mi-crobes, and incubated in solution for 134 d. In general, only 14 to33% of the DOC in forest floor, soil solution, and stream samplesdecomposed during the incubation. The relative order of average de-composition of DOC from the various strata was, from fastest to slow-est: throughfall, Oi horizon (forest floor), Oa horizon (forest floor),B horizon, stream, AB horizon, isolated fulvic acid, and upper Ahorizon. In short, biodegradability of DOM in the ecosystem profiledeclined vertically from throughfall to the A horizon and then in-creased with depth. The DON generally did not decay faster than theDOC — results consistent with the idea that hydrolysis of organic Nis linked to mineralization of DOC rather than occurring selectivelyin response to the biochemical need for N. Throughfall DOM couldbe decomposed during its passage through the upper soil, but decom-position seems too slow to be responsible for the bulk of removal ofDON and DOC that occurs in the mineral soil. Adsorption, ratherthan biodegradation, is more likely responsible for maintaining lowDOC substrate concentrations in the mineral soil and preventing itsloss into stream water.

THE BIODEGRADABILITY of xenobiotic compoundsin soil and water has been the focus of much

research. The biodegradability of naturally occurringDOM has received less attention, though there hasbeen considerable research on the fate of fresh autumnleaf litter leachate and on the ability of stream andlake DOM to support the growth of bacteria in thoseenvironments (Cummins et al., 1972; Lock and Hynes,1976; McDowell, 1985). Most of that research hasbeen used to advance the idea that natural DOM sup-ports significant bacterial growth (Meyer et al., 1987;Dahm, 1981; Bott et al., 1984). At the same time,there is a widespread belief that dissolved humic sub-stances, which make up much of natural DOM, arequite resistant to decomposition.

For obvious reasons, DOM in soil has received muchless attention than that in lakes and streams. The greatbulk of organic matter in soil is solid and is a muchlarger potential resource for soil microorganisms. Wewant to consider another aspect; however, what are

R.G. Quails, School of Forestry and Environmental Studies, DukeUniv., Durham, NC 27706; and B.L. Haines, Botany Dep., Univ.of Georgia, Athens, GA 30602. Contribution from the Instituteof Ecology and the Botany Dep., Univ. of Georgia. Supported byNational Science Foundation Grants BSR-8501424, 8514328, and9011661, and the Botany Dep. Palfrey Fund. Received 20 Feb.1991. 'Corresponding author.

Published in Soil Sci. Soc. Am. J. 56:578-586 (1992).

the processes that remove the DOM, and the nutrientsit carries, from soil solution and prevent its loss fromthe ecosystem? The DOM leached from the soil canalso potentially influence microbial processes indownstream lakes and streams.

In many respects, the biodegradation of the DOMis more important in its role in the N cycle than in theC cycle. In the forest watershed we studied at theCoweeta Hydrologic Laboratory in North Carolina, atleast 80% of the total N leached through the soil andinto the stream water was in the organic form (Quails,1989). Sollins and McCorison (1981) pointed out thatDON was by far the major form of N exported frommost forested watersheds. Nevertheless, at Coweeta,the DON underwent a 50-fold reduction in concentra-tion as it percolated from the upper soil horizons tothe stream. The mechanisms responsible for removingthis DON — whether biodegradation or adsorption —are preventing a long-term net loss of N from theecosystem and a subsequent input to downstream sys-tems.

An ideal assay of relative biodegradability of nat-ural DOM might have the following characteristics:(i) a substrate concentration characteristic of the en-vironment, (ii) the same concentration for differentsamples, (iii) a microbial inoculum representative ofall environments from which the samples were taken,(iv) an inoculum preconditioned to avoid a lag phase,(v) other factors potentially limiting decomposition ratessimilar between samples, or sufficient so that theywould not be limiting, (vi) prevention of the recyclingof organic C and N into the dissolved phase wherethey would be confused with the original substrates,(vii) standards of comparison, such as glucose and arefractory substance, as a control for unexpected toxiceffects and variation in inoculum, and (viii) a meansof distinguishing toxicity of one fraction of the mix-ture from the inherently low biodegradability of otherfractions.

Unfortunately, not all of these ideal criteria can bereconciled when dealing with complex mixtures fromenvironments where concentrations vary widely. Wecompromised uniformity for naturalness, in some cases,to meet as many criteria as possible.

Our objectives were to: (i) compare the relativebiodegradability of DOM from throughfall, soil so-lution from the Oi, Oa, upper A, AB, and B horizons,and stream water; (ii) determine whether DON is min-eralized faster than DOC; (iii) obtain at least a gen-eral, maximal measure of the rate of biodegradationto evaluate the hypothesis that biodegradation plays arelatively minor role in the removal of DOM com-pared with adsorption; (iv) distinguish whether two ormore very distinct fractions that differ greatly in biod-egradability occur in the mixtures of DOM, and (v)relate the relative biodegradability to differences in the

Abbreviations: DOM, dissolved organic matter; DOC, dissolvedorganic carbon; DON, dissolved organic nitrogen.

578

QUALLS & HAINES: BIODEGRADABILITY OF DISSOLVED ORGANIC MATTER 579

contents of humic substances and other componentsof the DOM.

METHODS

Sample Collection

Samples were gathered from 12 plots located in an oak-hickory forested watershed (WS-2) at the Coweeta Hydro-logic Laboratory in the southern Appalachian Mountains ofNorth Carolina. Elevations of WS-2 range from 700 to 1000m. Precipitation during the year of study, 1987, was 1537mm or 86.7% of normal. Plots were stratified by soil typeand slope position. Three soil types occur on the watershed:the Tusquitee series (a coarse-loamy, mixed, mesic UmbricDystrochrept) in the riparian zone, the Fannin series (a fine-loamy, micaceous, mesic Typic Hapludult) on the middleslopes and lower ridges, and the Chandler series (a coarse-loamy, micaceous, mesic Typic Dystrochrept) on the upperslopes and ridges. The forest floor is distinctly divided fromthe A horizon. It has distinct Oi and Oe horizons and, insome places, a distinct Oe horizon between them. Samplesof throughfall were collected with troughs. Water perco-lating from the bottom of the Oi and Oa horizons of theforest floor was collected in zero-tension soil water collec-tors (Jordan, 1968). Soil water was collected from the upperA horizon, the bottom of the AB or A2 horizons, the Bhorizon, and the upper C horizon in porous-cup vacuumcollectors at —50 kPa. Stream water was collected at thebase of the watershed. A more detailed description of thesite and sampling apparatus is given in Quails et al. (1991)(see also, Swank and Crossley, 1988).

Collections were made during 1-wk periods in February,May, August, October, November, and December 1988.Only samples from February, May, and August were as-sayed. Every effort was made to collect, filter, and preservethe samples as quickly as possible. Throughfall and forestfloor water samples were collected both during and within1 h after the end of rainstorms occurring within the sam-pling week. Soil water collectors were emptied every 24 hor less during the sampling period. Stream water was col-lected during periods of baseflow. Samples were placed onice within 1 h after collection, filtered through a WhatmanGF/F glass fiber filter (Whatman, Clifton, NJ) within 8 h,composited from all 12 plots, and frozen in liquid N2 within36 h in most cases. While conventional slow freezing maylead to flocculation in some samples (Giesy and Briese,1978), filtration through a glass fiber filter again after thaw-ing showed that there was no significant particle formationin our frozen samples.

Sample Preparation

To compare samples initially varying in DOC contentfrom 0.5 to 52 mg L'1, we diluted or concentrated allsamples to 6 mg L-1 so that the initial gross substrate levelwould be consistent. Samples with low DOC concentrationswere concentrated using a Virtis freeze concentrator (VirtisCo., Gardiner, NY) (Shapiro, 1961). This method was cho-sen because it is gentle and nonselective, and the sample isconcentrated at 0°C so microbial growth and evaporationof volatiles are prevented. The common DOC concentrationwas chosen because it was characteristic of upper A horizonsoil solution. The initial substrate concentration of individ-ual components, or enzyme-specific classes of compounds,undoubtedly varied, however.

Samples were all adjusted to pH 6 with NaOH or HCI.This pH was chosen because it was not low enough toinhibit decomposition and it was in the range of the streamand lower soil horizon samples.

Three other types of DOM were used for a comparison:

glucose, a freeze-dried fulvic acid sample isolated from ahumic lake (Quails and Johnson, 1983), and soluble organicmatter extracted from freshly fallen autumn leaves (Quailset al., 1991). Preliminary tests indicated that the glucosedecomposition was nutrient limited, so Na2HPO4, NH4C1,and NaNO3 were added to match the average total P andtotal N of the other samples. A mixture of other mineralsalts, designed to simulate soil solution, was also added.The same approach was taken for the fulvic acid, exceptthat the organic N and P content of the fulvic acid wastaken into account before additional N and P was added.The glucose and fulvic acid were added to low-DOC (0.4mg L-1) stream water along with the inorganic amend-ments.

A set of sterile controls utilizing the Oa horizon sampleswas also incubated to control for flocculation and vapori-zation of organics. The control solution was filter sterilized(0.2-(j,m filter) and aerated aseptically. Another set of con-trols consisting of deionized water, were inoculated andtreated as regular samples.

Inoculation

A section of forest floor and soil was dug up, kept in thelaboratory, and watered periodically to maintain a relativelyconsistent source of inoculum. A mixture of lower forestfloor material, A horizon soil, and stream sediment waschopped in a blender. The suspension was filtered sequen-tially through 37- and 0.2-u,m filters, and the particles be-tween 0.2- and =37-|xm diam. were washed with streamwater and resuspended. To speed initial microbial growthand prevent a lag phase, the suspension was incubated in amixture of various samples at 28 mg L- ' of DOC for 16h. The suspension was filtered and resuspended in streamwater, and 0.2 mL were added to each sample as quicklyas possible. An autoclaved and washed suspension was alsoadded aseptically to the sterile controls.

Incubation

We added 300 mL of each of the samples to each of threereplicate flasks. After inoculation, the flasks were aeratedwith charcoal-filtered, membrane-filtered, water-vapor-sat-urated air. The thin stream of air also gently circulatedsolution in the flasks. Two shredded-glass-fiber filters, whichcould be suspended and representatively sampled, were addedto provide extra surface area for microbial growth. Incu-bation temperature was maintained between 22 and 24 °C.

Five-milliliter samples were removed immediately afterinoculation, 1,2, 4, and 8 d later, and then at progressivelylonger intervals to 134 d. Suspensions were well mixedbefore removing the samples. Samples were filtered andanalyzed in duplicate for DOC using an automated persul-fate oxidation method (Model 700 TOC Analyzer, OI Corp.,College Station, TX). Subsamples from every second sam-pling time were analyzed for total dissolved N, NH4, andNO3, by methods detailed in Quails (1989). Dissolved or-ganic N was calculated from total dissolved N minus NH4and NO3.

Microbial biomass could generate secondary DOC or DONthat was not part of the original substrate via excretion, celllysis, or enzymatic hydrolysis of dead cells. To minimizethe accumulation of senescent and dead microbial biomasswe periodically removed a portion of the particulate matter.Every time a particular flask had lost an additional 10% ofits original DOC, 80% of the volume of the flask was fil-tered and then returned to the flask. Additional shredded-glass filters were added afterwards. This allowed 20% ofthe suspended biomass to serve as a base for continuedgrowth and presumably maintained a more actively growingbiomass. This periodic harvesting of particulate matter didnot seem to have any sustained influence on the shapes of

580 SOIL SCI. SOC. AM. J., VOL. 56, MARCH-APRIL 1992

the decay curves. There are no consistent inflections at 90,80, 70, or 60% DOC remaining in the decay curves (Fig.1). In many cases, the curves began to level out before anyfiltration of particles was done.

A budget of water volume removed and water remainingat the end of the experiment allowed evaporation to becalculated. Generally, = 12 mL out of the initial 1300 mLevaporated. We assumed a constant rate of evaporation andcorrected each of the analyses by a factor to compensatefor this evaporation.

Data Analysis and Assumptions

Because there usually seemed to be at least two relativelydistinct phases of decomposition during incubation, we it-eratively fit the curves of decomposition with time to a two-component first-order decay model (Wetzel and Manny,1972):

%DOC remaining = [(100 - fc)10-*»] + [blQ-ky] [1]

where t = time (units of d), 100 — b and b are the initialpercentages of the rapidly and slowly decaying compo-nents, respectively, and /:, and fc2

are tne rate constants ofthe two components. We estimated the initial proportionsof the relatively rapidly and slowly decaying componentsfrom the intercept of the slow portion of the decay curve.Since there were probably many components, the assump-tion that there were only two was a simplification, but thereseemed to be two relatively discrete categories of decayrates.

To avoid depending on the assumptions of first-orderdecay — the assumption being that decay rate was propor-tional to substrate concentration — we simply used theDOC remaining at the end of the incubation to draw mostconclusions about relative decay rates. We also attemptedto avoid depending on first-order assumptions by adjustingthe initial DOC to a common concentration. Since DOM isreally a mixture of substrates, however, this method wasnot entirely independent of these assumptions. The onlyconclusion that depended on the assumptions of the two-component decay model was our attempt to correlate therapidly decaying component with a specific DOC fraction.

Biodegradability and the Compositionof Dissolved Organic Carbon

We compared the decomposition of the DOC with itsinitial composition. We used a fractionation procedure thatdivides the DOC into humic substances, hydrophilic acids,phenols (i.e., weak hydrophobic acids), hydrophobic neu-trals, hydrophilic neutrals, and bases (Leenheer, 1981). Amore detailed description of this procedure and the resultsare reported in Quails and Haines (1991), but two additionalsamples were assayed for this study. We also assumed thatthe fractionation data for four samples gathered in Decem-ber were representative of our February samples used in thebiodegradation experiment, since the composition of thesoil DOC was very similar throughout the year (Quails andHaines, 1991). Samples gathered from the A and B hori-zons in May were not fractionated and were not includedin this comparison. Total carbohydrate content was mea-sured on selected samples and fractions using the phenol-H2SO4 assay (Handa, 1966).

We chose this fractionation procedure because three frac-tions contain substances that have traditionally been asso-ciated with varying degrees of biodegradability. The humicsubstances are believed to be refractory. The hydrophilicneutral fraction contains the free carbohydrates (those notbound to humic substances) (Leenheer, 1981), which havebeen generally regarded as one of the most biodegradablecomponents of DOM (e.g., McDowell, 1985). Polyphe-

nols, which are suspected of inhibiting microbial activity(Basaraba and Starkey, 1966), are found in the phenol frac-tion (Quails and Haines, 1991). In addition, the fractiona-tion procedure utilizes the interactions (water solubility andcharge at different pHs) that determine the physicochemicalmobility in the soil (Quails and Haines, 1991; Leenheer,1981).

RESULTS AND DISCUSSION

Relative Decomposition Rates

Decomposition curves show that only a small pro-portion of the DOM in most of the samples was rap-idly degraded (Fig. 1). Most DOM appeared to beresistant to decomposition, with the exception of thatin litter leachate and some throughfall samples. Thepercentage of initial DOC remaining during the in-cubation period is shown with a logarithmic scale onthe y axis to reveal any distinctly resolved phases ofexponential decay. Only a few of the samples exhib-ited any evidence of a lag phase in the first 4 d. Therelatively rapid phase of decomposition in the first 2wk consumed from 4.5 to 19% of the DOC of the soilwater and stream samples. In most samples this initialphase was relatively distinct, but the percentage ofDOC consumed was small. The August throughfallsamples, however, decomposed at a more or less grad-ually declining rate. The proportions of each compo-nent estimated by the two-component model are shownin Table 1.

There were significant changes in the biodegrada-bility of the DOM in the water as it percolated throughthe canopy, forest floor, soil, and finally into the stream.These changes are shown most clearly by the per-centage of initial DOC remaining at the end of theincubation period (Fig. 2). The statistically significantdifferences between the types of samples were shownby a multiple comparison of means from different strataand soil horizons (Table 2). For this analysis, we pooledvalues from the three different months. In addition, atwo-way analysis of variance on the throughfall, soil,and stream water results revealed the following: a sig-nificant difference among the main-effect means forsample type (P < 0.0001); borderline differences amongthe main-effect means for month (P = 0.06) and asignificant interaction of the effects of sample typeand month (P = 0.001).

The throughfall samples were clearly more biode-gradable than any of the others except glucose andleaf leachate. There were major differences betweenthe August and May throughfall samples, both of whichcontained dissolved organic matter leached from de-ciduous leaves during the growing season. Decom-position of the August throughfall sample was onlyslightly slower than decomposition of glucose duringthe first 4 d, but decomposition slowed thereafter (Fig.1).

The biodegradability of DOM in water decreasedsignificantly as it percolated downward through thecanopy and the Oi, Oa, and A horizons (Fig. 2, Table2). The A horizon samples were the least biodegrad-able of the samples and were comparable to the iso-lated fulvic acid samples. Within the mineral soil,there was a tendency for biodegradability of DOM toincrease with depth below the upper A horizon. Fi-


20 40 60 80 100 120 140 0100908070

60

5? 40

20 40 60 80 100 120 140 0100908070

60

50

20 40 80 100 120 140

40

20 40 60 80 100 120 140DAYS INCUBATED

250 20 40 60 80 100 120 140

DAYS INCUBATEDFig. 1. Decomposition curves for percentage of initial dissolved organic C (DOC) remaining plotted on a logarithmic y axis.

Sources are throughfall, soil solution, litter leachate, stream water, isolated fulvic acid, and glucose. Each point is the mean ofthree replicates. Note the generally slow decomposition of most samples and compare curves for DOC from different sources.

nally, the stream-water samples were similar to sam-ples from the AB, B, and Oa horizons. This similarityis not unreasonable, since stream-water DOM is acombination of DOM leached directly from organicdetritus in the stream channel and DOM that has passedthrough the soil.

Dissolved Organic Nitrogen

Partly because most samples lost only a small pro-portion of their organic C, there were few dramaticchanges in the C/N ratios (Table 3). Rather than show-

ing decomposition curves for the DON, it is morerevealing to express the results in terms of a changein the C/N ratio; that way we can confirm whether ornot DON-rich components were preferentially decom-posed. Not surprisingly, most samples that lost verylittle DOC did not change significantly in their C/Nratios. Only for the May throughfall was there evi-dence for preferential decomposition of DON-richcomponents. The opposite was true for the Augustthroughfall, the most biodegradable of all field sam-ples, in which there was a significantly lower C/Nratio after decomposition. It is possible that decom-


Table 1. Parameters of the two-component exponential decayequation fit to decay curves shown in Fig. 1: dissolved organicC remaining (%) = [(100 - b) 10-*a] where / = time (d),100 — b and b are the percentages of the rapidly and slowlydecaying components, respectively, and kt and k-, are therate constants of the two components.

Rapidly decaying

Source

Throughfall

Oi horizon

Oa horizon

A horizon

AB horizon

B horizon

Stream

Fulvicacid

LitterGlucose

Month

Feb.MayAug.Feb.MayFeb.MayAug.Feb.MayAug.Feb.MayAug.Feb.MayAug.MayAug.

—Nov.

—

100-6

2018491716

61319

5879

131316151315

73

59100

*,d-1

0.050.070.040.050.070.070.050.030.080.070.030.100.070.010.080.030.050.050.30.5

0.040.1

Slowly decayingb

8082518384948781959293918787848587859397

42ndi

kid-1 x I0"t

108

1058657533653635575

9nd

t Recorded numbers need to be multiplied by 10~4 for actual number.j nd = not determined.

100

Fig. 2. Final percentage of dissolved organic C (DOC) remainingafter incubation as a function of position in the verticalecosystem profile from which the sample was taken. Meansof three replicates for each month are plotted. Fulvic acidwas a standard of comparison. Note the general pattern ofdecreasing decomposition of DOC from throughfail downwardin the ecosystem profile to a minimum in the A horizon,with a slight increase in the B horizon.

position of the abundant carbohydrates in this sampleleft a more N-rich refractory component. In addition,the August Oa and the May AB samples had signifi-cantly lower C/N ratios after decomposition. In gen-eral, the DON was as refractory as the DOC.

McGill and Cole (1981) hypothesized that soil de-composers generally did not selectively hydrolyze N-

Table 2. Multiple comparison of means for the final percentageof dissolved organic C (DOC) remaining after incubation insamples from different horizons or strata.

Source of DOC

A horizonFulvic acidAB horizonStreamB horizonOa horizonOi horizonThroughfall

Mean DOCremaining

%86.1at81.7ab77.1cb74.2cd73.9cd70.7ed66.9e51.3f

t All observations for a particular horizon or stratum were grouped andsubjected to Duncan's multiple-range test (SAS Institsute, 1985). Meanswith the same letter are not significantly different at the 0.05 probabilitylevel.

Table 3. Initial and final C/N ratios for each type of sampleincubated.

Source of DOCThroughfall

Oi horizon

Oa horizon

A horizon

AB horizon

B horizon

Stream

Fulvic acid

Month (1987)

Feb.MayAug.Feb.MayFeb.MayAug.Feb.MayAug.Feb.MayAug.Feb.MayAug.MayAug.

—

Initial C/N

46.8(1.9)t47.5(1.5)*36.5(0.9)*41.7(2.9)42.4(2.4)43.0(1.7)54.5(0.2)45.9(1.0)*30.4(1.8)42.9(3.7)40.2(1.2)41.0(0.7)45.6(6.1)*39.3(2.4)40.1(0.8)30.2(1.2)NDt16.7(1.5)27.1(0.9)29.6(1.9)

Final C/N52.5(3.1)57.6(2.3)*25.3(4.8)*42.7(7.0)40.5(3.8)44.5(1.5)48.3(3.8)34.0(1.6)*34.3(2.8)43.7(1.4)42.2(1.6)42.6(2.6)34.8(3.7)*42.5(3.2)37.8(2.5)33.3(1.7)ND20.1(3.4)23.9(1.9)31.2(2.2)

* Pairs of means in the same row (initial vs. final) significantly different at0.05 probability level. The Mests were used to compare the initial andfinal C/N ratios (after arcsine transformation) (SAS Institute, 1985).

t Standard error for n = 3 replicates is shown in parentheses.t ND = not determined.

containing compounds in response to N limitation, butinstead N mineralization depended on C mineraliza-tion. Our results for soil DOM are generally consistentwith this hypothesis, since a selective hydrolysis oforganic N would tend to increase the C/N ratio of theremaining organic substrate. There are two biologicalprocesses that might result in a higher C/N ratio in theremaining substrate: (i) enzymatic cleavage of N-con-taining functional groups from the rest of a molecule,and (ii) selective decomposition of whole N-rich mol-ecules within the mixture of substances comprising theDOM. Even Process (ii) might not necessarily be in-duced by a biochemical "need" for N, but might oc-cur because the carbonaceous component of N-richmolecules are more easily decomposed. We saw noevidence of either process. The fact that N is widelydistributed among a variety of classes of compoundsin DOM (Quails and Haines, 1991) may partly explainwhy we did not see evidence of Process (ii).

The C/N ratios of forest floor DOM were compa-rable to or greater than those of solid Oi horizon litter,


80

tratt

o

60-

40-

20-

• THROUGHFALLo Oa HORIZONn A, AB HORIZONS• B HORIZONN STREAMA LITTER LEACHATE'

70

y=-1.27 x +85.5r=0.51

30 40 50 60% INITIAL DOC IN HUMIC SUBSTANCES FRACTION

Fig. 3. The percentage of dissolved organic C (DOC) lost overthe entire 137-d incubation period vs. the percentage of theinitial DOC classified as humic substances. Each pointrepresents the mean value for a particular source and month.The regression was significant (P < 0.05), but the correlation(r) was poor, indicating that the content of humic substancesnot the only factor explaining the decomposition rate of DOCfrom various sources and months.

and C/N ratios of A horizon DOM were higher thanA horizon solid organic matter (Quails et al., 1991).Thus, the DOM was not a richer source of N thansolid soil organic matter.

Composition of the Dissolved Organic Matter

We evaluated four hypotheses to explain the dif-ference in decomposition of the DOM samples:

1. Decomposition was inversely related to the con-tent of the humic substances.

2. Decomposition was related to the initial contentof the carbohydrate-rich (about 51%) hydro-philic neutral fraction.

3. Decomposition was inversely related to the con-tent of possibly inhibitory polyphenols.

4. The relative decomposition was related to theinitial organic N or P in the solutions.

Humic substances in water are widely believed tobe resistant to decay. Our isolated fulvic acid decayedvery slowly, as expected. We found, however, onlya very weak inverse relationship between decompo-sition rate and the initial percentage of the DOC class-ified as humic substances (Fig. 3). If throughfall andlitter leachate are excluded from the regression, thecorrelation is not significant. We believe this corre-lation was weak because there were also other classesof DOM besides humic substances that decayed slowlybut were not tightly correlated with the content ofhumic substances. For example, in the upper A ho-rizon samples, =42% of the DOC was humic, yet> 80% of the DOC remained undecomposed. A frac-tion classified as hydrophilic acids (comprising 9-34%of the DOC) was also colored and might contain somehumic-like substances (Quails and Haines, 1991). Sincehydrophilic acids constituted the bulk of the nonhumicDOC in most samples, at least part of this hydrophilicacid fraction must be resistant to decomposition toaccount for the amount of DOC that remained at theend of the incubation.

trmt

fc2ooQ

eo-

50-

40-

30-

20-

10

y=1.62x + 2.44r=0.83

» THROUGHFALLo Oa HORIZONn A, AB HORIZON• B HORIZONK STREAMA LITTER LEACHATE

10 20 30 40% INITIAL DOC IN HYDROPHILIC NEUTRAL FRACTION

Fig. 4. The percentage of dissolved organic C (DOC) lost duringthe 137-d incubation period vs. the percentage of the initialDOC classified as hydrophilic neutral substances. Each pointrepresents the mean value for a particular source and month.The regression was significant (P < 0.05).

60

40"

Q 20-

y= 1.60 x-10.2r=0.88

• THROUGHFALLo Oa HORIZOND A. AB HORIZON• B HORIZONN STREAM• LITTER LEACHATE

10 20 30 40% INITIAL DOC IN HYDROPHILIC NEUTRAL FRACTION

Fig. 5. The initial content of the rapidly decaying component(expressed as a percentage of the initial dissolved organiccarbon [DOC]) vs. the percentage of the initial DOC classifiedas hydrophilic neutral substances. The initial content of therapidly decaying component was estimated by fitting thedecay curves to a two-component first-order decay modelEach point represents the mean value for a particular sourceand month. The regression was significant (P < 0.05).

There was a much better positive correlation be-tween the percentage of DOC lost during the incu-bation period and the initial content of hydrophilicneutral substances (r = 0.83) (Fig. 4). These sub-stances may correspond with the generally small com-ponent of the DOC that decomposed relatively rapidlyin the first few weeks of incubation. In fact, the cor-relation (r) unproved to 0.88 when we estimated theinitial content of the more rapidly decaying compo-nent (using the biphasic decay model), and plotted itagainst the content of hydrophilic neutral substances(Fig. 5). Also, the percentages of the two componentscorresponded better, although quite a bit of variabilityremained unexplained. The hydrophilic neutral frac-tion averaged =51% carbohydrate content. Carbohy-drate content has often been used as an indication ofthe biodegradability of DOM. However, the gross car-bohydrate content should be interpreted with caution.


For example, the humic substances in our samplescontained an average of 16% carbohydrate. Sweet andPerdue (1982) also found that a large portion of thetotal carbohydrates in river DOM was bound to humicsubstances.

About 36% of the DOC extracted from freshly fallenautumn leaves was in the hydrophilic neutral fraction,about half of which was carbohydrate. This fresh au-tumn leachate has frequently been used in studies ofmicrobial growth on DOC (Dahm, 1981; Bott et al.,1984). It was clearly so much more biodegradable anddifferent in composition from the DOM leaching fromboth the forest floor and that found in stream waterduring most of the year (Quails et al., 1991) that webelieve it is not an appropriate model for DOM ingeneral.

There was no significant relationship between thepercentage of the initial DOC in the phenol fractionand the decomposition of DOC (P > 0.10). This phenolfraction was defined as weak hydrophobic acids byLeenheer (1981). The fraction contains polyphenols,such as tannins and flavonoids that have less thanabout one carboxylic acid group per 13 C atoms(Thurman, 1985; Quails and Haines, 1991), that aresuspected of inhibiting decomposition. In fact, the twosamples with the highest content of the phenol fraction(the August throughfall and the litter leachate) decom-posed fastest. In this biodegradation assay, it was im-possible to separate strictly the effects of inherentlypoor substrate quality from the inhibitory effect ofsome toxic fraction. This may be a moot point sincethe mixtures occur together in the soil solution.

In some cases, inorganic N has been found to becorrelated with the decomposition rate of fresh leaflitter (Meyer and Johnson, 1983). We found no sig-nificant correlation, however, between the initial in-organic N (not shown, n = 0.03, P > 0.10) or inorganicP concentrations (not shown, r = 0.02, P > 0.10) inthe solutions and decomposition of the DOM. Theglucose and fulvic acid solutions were excluded fromthe regressions.

Role of Decomposition of Dissolved Organic Matterin the Soil

To what extent can the rates of decomposition ob-served in this assay reflect the rates in the environ-ment? Although our experiments were mainly designedto measure relative biodegradability, we believe somegeneralizations can be made. The very slow decom-position of most of the soil-solution and stream-waterDOM samples under generally favorable laboratoryconditions suggests that most of this DOM is refrac-tory. Certainly the inoculation was sufficient to initi-ate rapid decomposition of the glucose with no apparentlag phase. Although high initial populations of bac-teria can completely consume small additions of glu-cose even more rapidly in culture, the initial glucoseconcentration was very low, only 6 mg L"1 of DOC.

Proving that a substrate or group of substrates isnot easily degraded requires proof by exhaustion. Mostsubstances can be degraded by some microorganismsunder at least some, perhaps peculiar, conditions. Un-less the culture conditions perfectly duplicate those inthe soil solution, we cannot be sure that the decom-

position rate is similar in soil. If we mimicked soilconditions, we would sacrifice the ability to compareDOM from different environments under uniform con-ditions. One problem with the laboratory incubationof solutions was that it was unlikely that the basidi-omycetes and some other multicellular fungi were ac-tive. These fungi may play a role in decomposition ofhumic substances. Aquatic bacteria that readily growin culture, however, can also degrade humic sub-stances (de Haan, 1972; Rifai and Bertru, 1980). De-spite the artificial conditions of incubations, most ofthe soil DOM probably cannot be considered labile.

Most of the soil-water and stream-water decompo-sition curves had a small, initially fast phase, sug-gesting that small components of the mixtures wererelatively labile (Fig. 1, Table 1). These small com-ponents could be extensively degraded during theirresidence times in the individual soil horizons. Thestream samples also exhibited a small, relatively labilecomponent — especially the May sample, where itamounted to =15% of the DOC. It seems doubtfulthat this component is extensively decomposed duringthe short residence time of stream water within thestreambed. The substrate is present at 10-fold lowerconcentrations in stream water, in which case the rel-atively labile fraction would be only ==0.1 mg L~'DOC, and temperatures are usually cooler than in thelaboratory. Nevertheless, a continuous flow of thislow-concentration substrate over attached bacteria onthe stones in the stream could conceivably supportsome oligotrophic bacterial growth. Meyer et al. (1987)used concentrated DOM, fractionated into molecularsize classes, from another similar stream at the Cow-eeta Hydrologic Laboratory in an experiment on bac-terial growth on DOM. The DOM was incubated for72 h at a considerably higher concentration than weused in our experiments. The decomposition rate wasrelatively fast during this initial period. The portionof the DOC that supported relatively rapid bacterialgrowth may have been analogous to our small rela-tively labile fraction. In addition, the much lower con-centrations we used could have resulted in much slowerbacterial growth rates. Meyer et al. (1987) also noteda strong association between apparent molecular sizeand biodegradability. In their experiment, none of thefraction > 10000 nominal molecular weight was con-sumed, while 86% of the less abundant fraction thatwas < 1000 nominal molecular weight was consumed.

Removal of Dissolved Organic Matter from the SoilProfile: Decomposition vs. Adsorption

We found no compelling evidence to reject the hy-pothesis that decomposition plays a relatively minorrole in the 100-fold reduction in DOC and the 50-foldreduction in DON (Quails, 1989) in the hydrologicprofile of the forest. Removal of DOM by adsorptionoccurs in minutes (Quails and Haines, 1992). Ad-sorption can explain the relatively constant DOC con-centrations at a particular place in the soil during stormsand throughout the year (Quails, 1989). Surely, ifbiodegradation controlled DOC concentrations, therewould be greater temporal variation.

Our results showed that only a small proportion ofthe forest floor, soil, and stream-water DOM decom-


posed rapidly, whereas a larger portion of throughfallwas labile. Whether the more labile fraction was sub-ject to the same degree of rapid adsorption we do notknow, but we do know that the DOM of the A horizonwas the most refractory. This suggests that eitherbiodegradation or selective adsorption ion the A ho-rizon or the forest floor selectively consumed the small,relatively labile fraction.

Adsorption in the A horizon was probably respon-sible for removing most of the DOC draining fromthe forest floor, reducing average DOC concentrationsfrom 33 mg L-1 (Quails et al., 1991) to ~6 to 8 mgL"1 in the upper A horizon. Adsorption also seems tomaintain a relatively constant DOC concentration at aparticular place in the A horizon regardless of waterflux or season (Quails and Haines, 1987, unpublisheddata). The A horizon DOM also seemed refractoryregardless of the residence time of the soil solution inthe A horizon. For example, during our sampling weekin May, strong storms had quickly flushed DOM fromthe forest floor into and through the A horizon. In theFebruary sampling period, however, residence time ofwater in the A horizon was much longer, but the soilDOM collected during both periods was refractory.

Some very slow rate of decomposition of even therefractory DOM probably occurs in the A horizon.Because a relatively constant DOM concentration sim-ilar to that used in our experiments is maintained insoil solution, decomposers are continuously exposedto this substrate.

The increase in biodegradability in the lower soilhorizons might have several explanations: (i) theremight be selective removal of the more refractory frac-tions by adsorption, (ii) there might be generation ofmore labile substances below the A horizon from roots,decomposition of solid soil organic matter, or micro-bial exudation, or (iii) the much lower concentrationsof DOM and the lower level of microbial activity inthe lower soil might act to decrease the rate of decom-position of the small, more labile fractions. Expla-nations (ii) and (iii) are not entirely consistent withthe reduction in biodegradability as forest-floor DOMmoved into the A horizon. Explanation (i), however,seems likely because there was a corresponding trendin the percentage of the DOC in the hydrophilic neu-tral fraction (Quails and Haines, 1991) that we suspectto be the more labile fraction and to be the one thatis less strongly adsorbed.

The emphasis of this work is on decomposition ofsoil DOM. Unfortunately, there has been very littleresearch on the subject so we must turn to studies ofstream-water DOM to compare our results. A numberof limnologists have attributed the removal of DOMfrom stream water and stream microcosms to micro-bial metabolism (Cummins et al., 1972; Lock andHynes, 1976; Dahm, 1981; Bott et al., 1984). Often,the source of DOM for these experiments is freshleachate from autumn-shed leaves. There may be sev-eral problems in associating the removal of DOM withmicrobial metabolism of leaf leachate: (i) removal mightrepresent adsorption as well as metabolism, (ii) ster-ilized sediments used as a control may release DOC(discussed by McDowell, 1985), (iii) the leachate offreshly fallen autumn leaves may be quite different

than the DOM entering streams most of the year, or(iv) the DOC concentrations used in experiments maybe unrealistically elevated, allowing microbial uptaketo be greatly accelerated (although adsorption may alsobe increased). McDowell (1985) found that 12.7% ofthe fresh leaf leachate he used was monomeric car-bohydrates, which might be expected to decomposerapidly. Meyer et al. (1987) compared fresh oak andhickory leachate to ambient stream and river DOMand found the degradation rate of the leachate to behigher in some molecular size fractions, so leachatemay not be representative of DOM in general.

McDowell (1985) found several forms of evidencethat the rapid removal of leaf leachate added to a streamwas mainly due to abiotic adsorption to sediments. Inlaboratory incubations, autoclaved sediments removedDOC almost as effectively as untreated sediments. A12% increase in sediment respiration after leachateaddition could not have accounted for the DOC re-moval. In the stream, he found that both large- andsmall-molecular-size DOC was rapidly removed. Therewas also no preferential removal of either monomericor polymeric carbohydrates relative to phenolics. Therewas a slightly greater uptake of polymeric carbohy-drates compared with unidentified DOC. He alsopointed out that addition of FeCl2 increased removalof DOC by sediments. In contrast, Dahm (1981) testedbiotic vs. abiotic uptake of fresh alder leachate andfound that, of the DOC removed, 20% was adsorbedand 77% was removed by microbes. The initial DOCwas elevated to =15 mg L"1.

Our results suggest the following hypothetical viewof the major relationships between DOM, adsorption,and microbial decomposition. Large quantities of DOMare generated in the forest floor and, to a much smallerextent, in the forest canopy. Most of this DOM is veryrapidly adsorbed in the A horizon (Quails and Haines,1992), where a certain proportion remains in the soilsolution by equilibrium with the adsorption sites. Bio-logical decomposition of organic matter in the dis-solved phase is too slow to remove a large portion ofthe DOM percolating through the soil. Adsorbed tothe soil matrix, this once-dissolved material is retainedand concentrated. Bacteria and fungi close to thesesurfaces exude exoenzymes that can hydrolyze the ad-sorbed materials in a much more concentrated form.This same argument may apply to the surface of streamsediments (McDowell, 1985). Over long periods oftime, the slow biological decomposition of soil or-ganic matter clears the adsorbing surface of solid or-ganic matter and the adsorption capacity is renewed.Thus, we view the fate of most soil DOM as a two-step process involving the initial rapid adsorption fol-lowed by slow biological mineralization.

ACKNOWLEDGMENTS

We gratefully acknowledge the technical assistance ofKent Tankersley with the incubations, the use of the TOCanalyzer at the Institute of Ecology, and partial financialsupport from NSF Grants BSR 85-01424, BSR 85-14328,and BSR 90-11661, and the Botany Dep. Palfrey Fund.

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Identification of Nutritional Influences on Cone Production

in Fraser Fir

Roger J. Arnold, J. B. Jett, and H. L. Allen

ABSTRACTFraser fir \Abies fraseri (Pursh) Poir.] is highly valued as a fresh-

cut Christmas tree. Commercial cultivation is limited partly by seedscarcity. The purpose of this study was to develop foliar tissue nutrientnorms for female cone yield to use in the Diagnosis and Recommen-dation Integrated System (DRIS), for ultimately improving cone yields.Two sets of these norms were developed, one set based on Februaryand the other on July foliar nutrient levels. Neither single mineral-nutrient concentrations nor other assessed tree parameters correlatedwith cone yield. However, nutritional discrimination between high-aud low-yielding trees was obtained with July tissue. Discriminationwith February needle samples was poor. Reasonable agreement wasobtained for orders of nutrient limitations diagnosed from the twoseasonally specific sets of norms. Results indicated the potential to useDRIS to aid in selection of clones and prescribing treatments to en-hance cone yields.

FRASER FIR is a small- to medium-size tree occur-ring naturally only in limited high-elevation lo-

Department of Forestry, North Carolina State Univ., Box 8002,Raleigh, NC 27695-8002. Received 16 Nov. 1990. 'Correspond-ing author.

Published in Soil Sci. Soc. Am. J. 56:586-591 (1992).

cations of North Carolina, Tennessee, and Virginia(Radford et al., 1983). It is highly valued as a fresh-cut Christmas tree, particularly in the eastern USA,where cultivation of Fraser fir is a major industry.

Seed production of quality fir seed is currently re-stricted by limited seed-production areas. Periodicityof cone crops is observed in Fraser and other firs,which complete the reproductive cycle from anthesisto cone maturation all within one growing season.Such patterns may arise from patterns of demand fornutrients and assimilates within trees during the grow-ing season (Powell, 1977). Reproductive bud differ-entiation coincides with rapid vegetative growth andcone development, both of which place high demandson available growth factors. During seasons whereboth rapid active growth and development of largenumbers of cones occur, lower levels of nutrients andassimilates tend to occur at the critical time of conebud initiation. This situation may favor vegetative de-velopment, latency, or even abortion of axillary buds(Powell, 1977; Sachs, 1977).

Cone productivity of individual trees in any partic-ular year is related to the proportion of the axillarybuds initiated that differentiated into cone buds theprevious year. Bud differentiation is influenced by the

biodegradability of dissolved organic matter in...

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