Relationship between microbial activity, biomass and organosulfur formation in forest soil

Soil Bid. Biochem. Vol. 25, No. 1, pp. 3339, Printed in Great Britain. All rights reserved

1993

0038-07 I7/93 $6.00 + 0.00

Copyright 0 1993 Pergamon Press Ltd

RELATIONSHIP BETWEEN MICROBIAL ACTIVITY, BIOMASS AND ORGANOSULFUR FORMATION IN FOREST SOIL ANDREW R. AUTRY’ and JOHN W. FITZGERALD~* ‘Environmental Site Restoration Inc., 5203 South Royal Atlanta Drive, Tucker, GA 30084 and *Department of Microbiology, University of Georgia, Athens, GA 30602, U.S.A. (Accepted 25 July 1992) Summary-The capacity to form organic S was determined for microorganisms present in soil samples, collected from different depths of an eastern white pine forest. Inhibitors of eukaryotes (cycloheximide and amphotericin B), gram-negative prokaryotes (polymyxin B), and aerobically-respiring prokaryotes

and eukaryotes (sodium azide) were used to assess the relative contribution of each group to organosulfur formation over a wide range of added sulfate. Values for microbial biomass (direct counts and ATP content) and activity (soil respiration) were estimated and correlated with organosulfur formation potentials at various soil depths. Most of this latter activity, regardless of concentration of added sulfate, was mediated by aerobically respiring prokaryotes. In each horizon, however, increasing concentrations of sulfate induced a shift in the physiological types of microbial populations responsible for organic S formation. This is consistent with the observed multiphasic uptake kinetics for sulfate characteristic of samples collected from each horizon. ATP pool sizes and native soil respiration rates exhibited positive relationships with organosulfur formation when 7.5 nmol sulfate (r = 0.86, 0.81, respectively) and saturating concentrations of sulfate (r = 0.79, 0.72, respectively) were employed, separately. Amounts of added sulfate also had a positive effect on soil respiration rates (P < 0.0001). Collectively, the data suggest that exposure to sulfate stimulated endogenous aerobic respiration, generating ATP, that was used in part to form organic S.

formed; De Meio, 1975), indices of microbial activity and energy availability may provide a better estimate of the capacity of soil samples to form organic S. Incorporation of [3H]thymidine into bacterial DNA has been used extensively as an index of microbial activity in aquatic systems (Findlay et al., 1981) and soils (Christensen et al., 1989). Due to the severe limitations of this technique (Hollibaugh, 1988), however, it may not yield the best estimate of the potential capacity of a given soil to form organic S. Soil respiration, quantified by measuring CO2 evolution, has no such limitations, and has been used extensively to estimate soil microbial activity (see e.g. Dobbins and Pfaender, 1988). Moreover, because of the ATP-generating capacity that aerobic respiration provides, measurements of respiration also give an indirect index of the energy availability of a given soil. In addition, measurements of ATP pool sizes have been used to estimate both energy availability and microbial biomass (Jenkinson and Oades, 1979; Jenkinson et al., 1979). Whereas attempts have been made to correlate microbial population density and ATP pool sizes with xenobiotic degradation in groundwater (Jensen, 1989), no reports on the correlation of these parameters with S cycling have appeared. It was of interest therefore to determine which interactions, if any, existed between these indices and organosulfur formation capacities at progressively increasing depths within the soil profile.

INTRODUCTION

Organic S, in many cases the principal component of total S in forest soils, is generated through the incorporation of sulfate-S into organic matter (Strickland and Fitzgerald, 1985; Fitzgerald et al., 1985; Autry et al., 1990). Using a modification of the “heterotrophic activity method” of Wright and Hobbie, we demonstrated a sulfate concentration dependency for organosulfur formation (Autry and Fitzgerald, 1991a) with saturation kinetics persisting at all depths within the soil profile. Multiphasic kinetics for this process were observed for soil samples taken from a variety of forested sites, indicating that more than one microbial population was responsible for sulfate uptake (Autry and Fitzgerald, 1991a). It appears that the microbial populations responsible for sulfate incorporation within a given horizon are very diverse. This notion is supported by the findings of Sbrheim et al. (1989), in which bacteria randomly isolated from soils were found to be phenotypically diverse, and of Torsvik et al. (1990), who indicated a high diversity in the DNA content of soil bacteria. The community size of a given horizon, however, may not be directly related to the microbial activity within that horizon. Owing to the energy requirement for organic S formation (2 mol ATP per S linkage *Author for correspondence. 33

34

ANDREW R. AUTRY MATERIALS

and

AND METHODS

Sample collection

Samples were taken from the A (O-10 cm), B (IO-25 cm), BC (25-40 cm), and C (40-55 cm) horizons using a pit dug on Watershed 1 at the Coweeta Hydrologic Laboratory near Franklin, N.C. Eastern white pine (Pinus strobus) is the dominant overstory vegetation and soils are in the fine loamy Fannin series of Typic Hapludults. A detailed site description was provided by Swank and Crossley (1988). Samples were maintained, field-moist in sealed polyethylene bags at about 4°C. Organic S formation and mobilization capacities

Organic S formation capacities were determined by using the constant specific radioactivity approach (Autry and Fitzgerald, 1991b). Triplicate samples (1 g wet wt, not sieved) were kept at 20°C for 48 h with sulfate concentrations (mixture of 35S-labeled and unlabeled) ranging from 7.5 nmol to 400 pmol gg’ wet wt. The lower concentration is consistent with the sulfate deposition history for the study site. Specific activity of 8.33 x 10’ Bq mmol-’ was maintained throughout. The validity of this approach has also been assessed by isotope dilution analysis and both methods yielded statistically indistinguishable values for samples taken from all horizons of this watershed (Autry and Fitzgerald, 1991b). Following incubation, samples were extracted to yield water, salt, acid and base extracts. Soluble 35Swas determined in the water extracts, adsorbed 35S in the salt extracts and 35S incorporated into organic matter was calculated as the sum of that present in the acid and base extracts. Kinetic parameters for organic S formation were calculated by linear regression analysis of Wright-Hobbie data plots as described in detail previously (Autry and Fitzgerald, 1991a). Briefly, Vmaxis the reciprocal of the slope of the line resulting from these plots, whereas K1 (half-saturation constant) is the X-intercept less the amount of endogenous total sulfate. The size of the endogenous total sulfate pool was estimated by adding the amounts of soluble and adsorbed sulfate determined for a particular sample (see sulfur analysis). To assess the capacity of soil samples to mobilize (solubilize and mineralize) organic S (Strickland et al., 1984), samples (1 g wet wt, not sieved) were exposed in triplicate at 20°C for 24 h to a concentration of sulfate equal to that yielding saturation for organic S formation. The samples were then extracted with NaH,PO, to remove any 35S not incorporated into organic matter and washed with a 1: 5 soil:water solution in an attempt to replenish microflora lost during the NaH,PO, extraction. After exposure for an additional 24 h at 20°C the samples were extracted to yield salt, acid, and base fractions. Organic S mobilized during this latter incubation was calculated as the 35S present in the salt extract whereas nonmobilized organic S was calculated as the 3’s

JOHN W. FITZGERALD

present in the acid and base extracts. Mobilization capacities were calculated as a percentage of the organic S formed initially. Recoveries of added 35S exceeded 90% for both the formation and mobilization assays. Znfuence of antimicrobial agents

To assess which group of organisms was responsible for organic S formation at various sulfate concentrations, the following protocol was employed. Triplicate soil samples (1 g wet wt, not sieved) were amended with either filter-sterilized deionized water, sodium azide, a combination of cycloheximide and amphotericin B, amphotericin B alone, or polymyxin B alone. Final concentrations were several fold higher than those necessary for complete inhibition of test organisms and were chosen to achieve a maximum effect. These were 128, 2, 1, and 1OOpg g-r sample dry wt, respectively. Immediately following the addition of the inhibitors, samples were exposed to either 7.5 nmol, 50 or 380 pmol [35S]sulfate g-r wet wt. Specific activity of 8.33 x 10’ Bq mmol-’ was maintained throughout. Samples were then extracted and organic “S was quantified as above. Enumeration of bacteria

Cells were recovered from triplicate soil samples by shaking for 30 min with filter-sterilized (0.22 pm) 0.1% sodium pyrophosphate, pH 7.0. The resulting extracts were preserved with formaldehyde (final concentration 4%) and subjected to ultrasonication for intervals up to 4 min (Ellery and Schleyer, 1984). Sonication for longer intervals had no significant effect on cell counts (data not shown). Extracts were then diluted and filtered through polycarbonate (0.22 pm) that had been stained with Irgalan black (Nucleopore Corp.). Bacteria trapped on filters were stained with 0.01% a&dine orange and 20 fields were counted using an Olympus epifluorescence light microscope (Zimmerman and Meyer-Reil, 1974). ATP measurements

ATP was extracted from the soil by ultrasonication in a solution of trichloroacetic acid 0.5 M, Na,HPO, 0.25 M, and paraquat dichloride 0.1 M (Jenkinson and Oades, 1979). The superiority of this extraction technique over other methods, such as the bicarbonate-chloroform extraction, was demonstrated by Jenkinson et al. (1979). ATP was quantified using the firefly luciferin-luciferase enzyme system. Carbon determinations

Total C was determined using a Leco analyzer. Carbonate-C was liberated from soil as CO,. Following acidification with cold 2~ HCl, the gas was trapped in 1 M NaOH and determined titrimetrically (Kalembasa and Jenkinson, 1973). Organic C was calculated as the difference between total and carbonate C.

Organosulfur formation Respiration

studies

The influences of sulfate concentration and depth on soil respiration were assessed using a modification of the technique of Anderson (1982). Sulfate concentration and sample depth were the dose (independent) variables, and respiration rate was the response (dependent) variable. Briefly, triplicate samples (10 g wet wt) containing either 7.5 nmol or 50 pmol SOi- g-i wet wt were kept for 5 h in sealed 100 ml bottles. The volume of sulfate solution added was 200 ~1 g-i wet wt and, in the case of the control, deionized water was added in place of sulfate. All solutions were filtersterilized (0.22 pm membrane) prior to use. The CO, evolved was trapped in 1 M NaOH and quantified titrimetrically as above. Samples were exposed for 5 h rather than for the traditional 24 h because the shorter exposure yielded respiration rates that were linear with respect to time. Sulfur analysis

Total S was determined by hydriodic acid reduction following hypobromite oxidation (Autry et al., 1990). Soluble sulfate was extracted from soil by shaking for 15 min in a 1:5 soil: water mixture, followed by centrifugation. The supernatant was filtered (0.22 pm membrane) and stored at about 4°C prior to analysis. The residue was resuspended in 20 mu Na,HPO, (1:5 ratio) and shaken for 30 min, followed by centrifugation. This procedure was repeated, and the supernatants, containing adsorbed sulfate, were pooled, filtered, and stored at 4°C. Inorganic sulfate in each extract was quantified by anion chromatography (Autry et al., 1990). For purposes of the current study, organic S was calculated simply as the difference between total S and total sulfate. It should be noted, however, that constituents of this fraction have been defined and quantified in a variety of litter and soil samples from this watershed and elsewhere (Fitzgerald et al., 1985; Autry et al., 1990). Unlike some aquatic systems, the only major form of inorganic S in this and most forest soils is SO:-. Attempts to detect S2- or f&O:-, even in trace quantities, have been unsuccessful (StankoGolden, unpubl.). Statistical

analysis

Data derived from the kinetic studies was subjected to one-way analysis of variance and Duncan’s MulTable 1. Kinetic parameters

for organic

S formation

35

tiple Range Test. Linearized data was subjected to simple linear regression analysis. Data from direct counts of bacteria, sulfur analysis, inhibitor studies and ATP determinations were subjected to one-way analysis of variance and Duncan’s Multiple Range Test. Data derived from the respiration experiment were subjected to 2-way ANOVA and Duncan’s Multiple Range Test within a treatment level. The significance level for all multiple comparisons was tl = 0.05. RESULTS

Kinetic parameters for organic S formation and mobilization are summarized in Table 1. Values for the maximal rate of organic S formation (V,,,) increased with increasing soil depth. The concentration of added sulfate required for saturation (calculated by doubling the half-saturation constant, K,) also increased with sample depth. The lone exception was the B horizon, where a substantial decrease was observed. Unlike potentials for organic S formation at saturation, the amounts of organic S formed from environmentally relevant amounts of sulfate (e.g. 7.5 nmol) decreased with increasing depth. Moreover, when this amount of sulfate was used to generate organic S, the amounts of organic S mobilized also decreased with depth. Conversely there was a general increase with depth in the amount mobilized at saturation. Although the percentage of organic S mobilized was unaffected by sulfate concentration, organic S mobilization decreased with depth and ranged from 81% in the A horizon to 64% in the C horizon. The effect of various antimicrobial agents on organic S formation by samples exposed to different sulfate additions is summarized in Table 2. Irrespective of depth and amount of sulfate added, sodium azide caused the greatest inhibition of organic S formation (>45%). The influence of other agents varied with depth and sulfate concentration. With surface samples (A horizon), the inhibition of organic S formation caused by the combined addition of cycloheximide and amphotericin B and amphotericin B alone were not significantly different (ANOVA, Duncan’s Multiple Range Test, CL= 0.05). Furthermore, these values were not significantly different from those of the control when 50 or 380 pmol SOi-

and mobilization

in samples collected

from various

Organic S formeds (pmol S g-’ dry wt 48 h-‘)

Horizon’

Depth (cm)

Apparent If,.. (pmol S g-l dry wt h-‘)

A B BC C

O-10 lo-25 25-40 40-55

0.22 0.25 0.30 0.33

Sulfate concentration yielding saturation (pmol g-’ dry wt)t

At saturation

213.4 171.6 241.2 303.1

*All data for these horizons IS lower limit, due to multiphasic kinetics. tCalculated as K, x 2. SResults expressed as means with n = 3. Standard error < 15% in all cases

10.6 12.1 14.2 15.7

From 7.5 nmol Sq2.3 2-2 2.1 1.7

x x x x

IO-’ 10-J 10-j 10-3

forest soil horuons

Organic S mobilizedt (gmol S g-’ dry wt 48 h-‘)

At saturatlon 8.6 8.9 9.1 10.0

From 7.5 nmol SO:1.9 1.6 1.3 I.1

x x x x

10-X IO-’ 10-j 10-3

36

ANDREW

R. AUTRYand JOHN W. FITZGERALD

Table 2. Effect of various inhibitors on organic S formation capacities for various forest soil horizons* Percentage Inhibition of organic S formation from added sulfate concentrations (pmol g-’ dry wt) of: Addition

Horizon A

B

BC

C

None (Control) Sodium ande Polymyxin B Cyclohexnnide and Amphotericin B None (Control) Sodium azide Polymyxin B Cycloheximide and Amphotericin B None (Control) Sodium azide Polymyxin B Cycloheximide and Amphotericin B None (Control) Sodnun azide Polymyxin B Cycloheximide and Amohotericin B

7.5 x 10-J

50

380

Ob 74c 266 22’ 13’ Ob 64c Ob 23b 18b Ob 72’ 5b 29d 24’ Ob 65’ Ob 356 35d

Ob 465 43’ 14b 16b Ob 77’ 76’ Ob 32d Ob 61’ 5b 12b 25b Ob 60’ 13b

Ob 57E 566 23b

amphotericin B

amphotencin

B

amphotericin B

amphotericin B

$ 48’ 22b 6b 16b Ob 49’ lgb 4b lgb Ob 67’ 4Od

*Results expressed as means with II = 3. Standard error < 15% m all cases. Values that were not significantly different (ANOVA, Duncan’s Multiple Range Test, tl = 0.05) within a sulfate treatment level within a horizon are designated by the same letter.

was added. However, this trend was not observed when 7.5 nmol SOi- was added. Also, the inhibition produced by polymyxin B was statistically indistinguishable from that caused by sodium azide when 50 or 380 pmol SOi- was used to generate organic S. With samples from the B horizon, inhibition of organic S formation caused by cycloheximide and amphotericin B or by amphotericin B alone were not significantly different from each other, from that caused by polymyxin B or from the unamended control when 7.5 nmol or 380 pmol SO:- was added. When 50 pmol SOi- was added, however, the inhibition caused by the cycloheximide-amphotericin B combination was significantly lower than that caused by amphotericin B alone, but the inhibitions arising from amendment with sodium azide and polymyxin B were not. With samples from the BC horizon, the inhibition caused by polymyxin B, cycloheximideamphotericin B, and amphotericin B alone were not significantly different from each other or from the unamended control when 50 or 380pmol SOi-

was added. After addition of 7.5 nmol SO:-, the reduction in activity due to cycloheximideamphotericin B did not significantly differ from that of amphotericin B alone, and the inhibition caused by polymyxin B did not differ significantly from the unamended control. With C horizon samples exposed to 7.5 nmol and 50 lmol SOi- the trends were the same as with the BC horizon samples. This was not the case with C-horizon samples containing 380 pmol SOi- (Table 2). Bacteria1 biomass, determined by direct counting, generally decreased with increasing depth (Table 3). However, there was a substantial increase in cell numbers in the B horizon. Values for ATP pool sizes followed an identical trend. Values for organic C declined sharply, and without exception, with increasing depth in the soil profile. Organic C was the primary form of C at all depths analyzed, accounting for >85% of total C (Table 3). With respect to soil respiration, the results were similar to those obtained for biomass. There was a

Table 3. Biomass and carbon content of various forest soil horizons Carbon content (mg g-’ dry wt) Horizon A B BC C

Bacterial biomass*,t (cells g-’ dry wt)

ATP content*$ (Irg g-l dry wt)

1.8 x 10” 1.3 x 10” 8.3 x IO* 1.8 X 108

4.66 6.16 2.29 1.10

Carbonate: 0.86 1.79 1.10 0.40

Organi& 27.32 16.07 9.33 3.89

*Corrected for extraction efficiency. tResults expressed as means with n = 180. Standard error ~8% in all cases fResults expressed as means with n = 3. Standard error ~20% in all cases. #Determined by difference.

Soil pH 4 66 5.67 5.70 4.87

Organosulfur formation Table 4. Influence of sulfate concentration resoiration rate

and horizon on soil

Rate (~cmol g-’ dry wt h-‘) at concentrations (pm01 g-’ dry wt) of: Horizon A B BC C

0

7.5 x 10-j

50

62.2* (9.7) 17.8 (7.9) 31.4 (9.3) 28.8

47.5 (12.0) 36.2 (7.9) 25.2 (5.9) 39.5

60.4 (8.4) 66.9 (10.1) 61.6 (9.7) 46.7

(1.8)

(1.8)

380 69.5 (4.8) 94.1 (4.8) 59.8 (1.9) 13.7 (7.8)

(1.8)

*Rates expressed as means with n = 3. Standard error is giveo in

significant decrease in rates for unamended samples, except for the B horizon (P < 0.0079). In general, respiration decreased by > 50% from the A to the C horizons (Table 4). As with biomass and ATP content, respiration rates for the unamended B horizon samples were significantly higher (P < 0.0001) than those of the other unamended horizons. An effect of depth on respiration rates was not observed with any of the sulfate amended samples (P < 0.40 in all cases) but was strongly expressed in the unamended control (P < 0.006). In general sulfate additions exerted a positive influence on respiration rates irrespective of horizon (P < 0.0001). This effect was evident with samples taken from the B, BC and C horizons (P < 0.005,0.049 and 0.004, respectively), but not for the A horizon (P < 0.4376). There was also significant interaction between depth and sulfate on respiration rates (P < 0.0304; Table 4). The pool sizes for total S and total organic S exhibited no obvious trend with increasing depth (Table 5). The content of adsorbed and total sulfate increased with increasing depth, and pool sizes were roughly 2.5 times greater in the C horizon than in the A horizon (Table 5). When considering the data collectively, several trends emerge. Bacterial cell numbers exhibited positive relationships with ATP pool sizes (r = 0.78) and native soil respiration rates (r = 0.76). Furthermore, ATP content was positively correlated to organic S formation, using both environmentally relevant (7.5 nmol; r = 0.86) and saturating sulfate concentrations (r = 0.79). Similarly, respiration rates were positively correlated to the amount of organic S formed from both 7.5 nmol (r = 0.81) and saturating sulfate concentrations (r = 0.77). Positive relation-

37

ships were also observed between adsorbed sulfate and organic S formation activity at both the lower amount of added sulfate (r = 0.70) and at saturation (I = 0.97). Total sulfate pool sizes, however, were only weakly correlated to organic S formation potentials at saturation (r = 0.61). A positive relationship was observed between soluble sulfate concentrations and respiration rates (r = 0.83). Negative relationships existed between organic C and organic S formed from 7.5 nmol sulfate (r = 0.72) and saturating sulfate concentrations (r = -0.98). All correlations were highly significant (P < 0.001). DISCUSSION

In light of the inhibition of organic S formation by sodium azide, it appears that aerobically respiring organisms are responsible for the bulk of this process, regardless of depth and sulfate concentration. This is in agreement with results of work with surface horizons where most of this activity was abolished following addition of this respiratory inhibitor. Strickland and Fitzgerald (1984) attributed the activity which remained to preformed enzymes present in the soil. While the use of metabolic inhibitors to assess the contributions of various groups of organisms to ecological processes has some methodological problems, chiefly resistance of many protozoa to cycloheximide (Sanders and Porter, 1986), the approach can still be used to gain a sense of the relative importance of each group. Collectively, the data suggest that prokaryotes were the microorganisms primarily responsible for organic S formation at all depths within the soil profile regardless of the concentration of added sulfate. This conclusion is based on the observation that cycloheximide and amphotericin B caused no significant inhibition of organic S formation in 9 of the 12 treatments. The exceptions were the A horizon soil exposed to 7.5 nmol sulfate, the B horizon soil exposed to 50pmol sulfate, and the C horizon soil exposed to 380 pmol sulfate. In none of these cases, however, did the inhibition caused by addition of the eukaryotic inhibitors exceed 35%. Attempts to further differentiate this eukaryotic activity into that mediated by protozoa and amphotericin B-resistant fungi or that mediated by amphotericin B-sensitive fungi, indicated that it was

Table 5. Sulfur status of various forest soil horizons* Sulfur component (fig S g-’ dry wt) Horizon

Total

Total organic

Soluble sulfate

Adsorbed sulfate

Total sulfate

A

215.1

Il

169.6 255.2

C

200.7

6.89 (3.2) 13.81 (8.1) 6.35 (2.5) 3.37 (1.7)

32.72 (15.3) 54.23 (32.0) 84.79 (33.2) 88.56 (44.1)

39.61 (18.5) 68.04

BC

175.5 (81.5)t 101.6 (59.9) 164.1 (64.3) 108.8 (54.2)

*Results expressed as means with n = 3. Standard error < 10% in all cases tTota1 S is givm in parentheses as a percentage.

‘?;‘d (35.7) 91.93 (45.8)

38

ANDREW

R.Aura~ and JOHNW.FITZGERALD

primarily the latter group that was responsible for organic S formation. This is similar to results obtained with samples from surface soil in which candicidin-sensitive fungi were implicated as the major eukaryotic contributors to this process (Strickland and Fitzgerald, 1984). However, it is apparent that in C horizon soils exposed to 380 pmol sulfate (saturating sulfate concentration), protozoa or amphotericin B-resistant fungi played some role in the incorporation of sulfate into organic matter. This conclusion is not without precedent. Gupta and Germida (1989) demonstrated that amoeba can have a significant role in S cycling in soil ecosystems. Within the prokaryotic component of organosulfur formation activity, it appears that Gram-positive bacteria were the primary mediators for all sulfate concentrations and depths examined. This is in agreement with Strickland and Fitzgerald (1984), who found Grampositive bacteria implicated as major contributors to this process in A horizon soils. Results of our work indicate that these bacteria accounted for the bulk of activity in all horizons exposed to 7.5 nmol sulfate, the B and BC horizon soil exposed to 380 pmol sulfate, and the BC and C horizon soil exposed to 50 pmol sulfate. Gram-negative bacteria were the organisms primarily responsible for organic S formation in the A horizon samples exposed to 50 and 380 pmol sulfate and in B horizon soil exposed to 50 ymol sulfate. These bacteria were implicated as a result of the significant inhibition of organic S formation by polymyxin B. In many cases the influence of this antibiotic did not differ significantly from that caused by azide. In every horizon analyzed, however, increasing concentrations of sulfate induced a shift in the primary populations responsible for organic S formation. For example, in the A horizon, Gram-positive bacteria were responsible for organic S formation at an environmentally-relevant sulfate concentration (7.5 nmol). However, as the sulfate concentration was increased to 50 and 380 pmol, Gram-negative bacteria became the predominant mediators of this process. Indices of soil microbial biomass (cell numbers and ATP content) generally followed the same trends with increasing sample depth. That is, with the exception of the B horizon, where a substantial increase was observed, soil microbial biomass tended to decrease with increasing depth. These findings agree with those of Cochran et al. (1989), in which sharp decreases in bacterial biomass with increasing depth were observed. The strong positive correlation between these variables (r = 0.78) was not surprising, as ATP is a well-recognized indicator of microbial biomass. Adhesion of bacteria to clay has been noted (Van Loosdrecht et al., 1989), and this may explain the high values for each parameter observed with B horizon samples. This genetic horizon generally has a high clay content and soil from the study site is no exception, In general, values for bacterial cell counts and ATP content agreed well with the amounts of

organic S formed from 7.5 nmol sulfate. However, there was little change in the amount of organic S formed in the B horizon samples despite a sharp increase in microbial biomass. Values for these indicators did not agree with potentials for organic S formation at saturation, which increased without exception with increasing depth. A possible explanation for this phenomenon is that bacterial populations at the lower depths have a greater capacity to form organic S (i.e higher V,,) at the higher concentrations of added sulfate. Furthermore, because the a&dine orange method yields counts which include both dead and living cells (Zimmerman and Meyer-Reil, 1974), the proportion of active bacteria responsible for organic S formation may be much larger in the lower horizons exposed to higher concentrations of sulfate than in the upper horizons exposed to the same sulfate concentrations. Thus, the relationships between organosulfur formation and native microbial populations at increasingly higher sulfate inputs may be more appropriately elucidated by examining indices of microbial activity rather than biomass. With the exception of samples from the B horizon, soil respiration rates decreased with increasing depth. These rates were significantly correlated to bacterial cell counts, ATP concentrations, and organic S formation capacities (both from 7.5 mnol sulfate and at saturation). These relationships were not surprising, owing to the large ATP generating capacity of aerobic respiration and the ATP requirement for organic S formation (De Meio, 1975; Strickland and Fitzgerald, 1985). Sulfate additions also exerted a significant positive influence on respiration rates when the data were considered collectively. This increase in respiration after exposure to sulfate indicates that the resident soil microorganisms are metabolically very flexible. Such metabolic flexibility has been noted for groundwater bacteria (Bengtssiin, 1989). On this basis, one would expect that the pool size of soluble sulfate, a readily available form of S, would be higher in the surface A horizon relative to the other, deeper horizons. This belief was not supported by results of this study, however. One possible explanation for this apparent contradiction is that the microbial populations responsible for organic S formation in the A horizon may be more efficient, requiring less added sulfate to achieve saturation, than are their lower horizon counterparts. This possibility was supported in part by the lower value of the collective half-saturation constant (K!) observed for the A horizon samples relative to BC and C horizons samples. Collectively, the data suggest that increasing inputs of sulfate, such as those encountered in acidic precipitation, stimulate aerobic respiration by microbial populations in the soil, generating ATP, which is then used to drive the organic S formation process, using the added sulfate as a source of S for incorporation into organic matter.

Organosulfur formation work was supported by a National Science Foundation Long Term Ecological Research Multi-investigator grant. We thank W. J. Wiebe and M. A. Moran for advice. A. R. Antry was supported in part by a University-wide fellowship awarded by the Graduate School.

Acknowledgements-This

39

Gupta V. S. S. R. and Germida J. J. (1989) Influence of bacterial-amoeba1 interactions on sulfur transformations in soil. Soil Biology & Biochemistry 21, 921-930. Hollibaugh J. T. (1988) Limitations of the [3H]thymidine method for estimating bacterial productivity due to thymidine metabolism. Marine Ecology Progress Series 43, 19-30.

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