Application of the heterotrophic activity method to organosulfur formation in forest soils

Soil Biol. Biochem. Vol. 22, No. 6, pp. 143-748, 19% Printed in Great Britain.All rightsreserved

0038-07 I7190 53.00 + 0.00

Copyright0 1990PergamonPressplc

APPLICATION OF THE HETEROTROPHIC ACTIVITY METHOD TO ORGANOSULFUR FORMATION IN FOREST SOILS ANDREW R. AU~Y

and JOHN W. FITZGERALD

Department of Microbiology and Institute of Ecology, University of Georgia, Athens GA 30602, U.S.A.

(Accepted IS April 1990) Summary-Maximum capacities of soil samples to incorporate ‘5S-labelled inorganic sulfate into organic matter were assessed over a wide range of added SOi- concentrations. The effect of sample depth on these capacities was also determined. Kinetic analysis was applied, and, with samples collected from a mixed fir-spruce forest site, capacities were found to decrease with increasing depth in the soil profile, from 5.13 to 4.18 pmol organic S formed g-’ dry weight 48 h-‘, in the E and C horizons, respectively. All horizons exhibited saturation of the capacity of the soil to form organic S. Multiphasic uptake kinetics for SO,were observed for the BC horizon soil. The validity of the application of the heterotrophic activity method to studying Se- incorporation in forest soil was confirmed by results of this study.

INTRODUCTION

To fully assess the deleterious effects of acidic deposition on forested ecosystems, a complete understanding of sulfur retention mechanisms in these ecosystems is necessary. Sulfate, the principal anionic component of acidic precipitation (Likens and Bormann, 1974) is extremely mobile in its soluble form, and if not immobilized by adsorption or incorporation into organic matter upon entering forests is subjected to leaching and eventual loss from the ecosystem in streamflow (Heute and McCall, 1984). Sulfate-leaching is associated with a concomitant loss of nutrient cations by virtue of electrostatic interaction (Cole and Johnson, 1977; Johnson, 1980). Immobilization and subsequent retention of SO:-, however, has been shown to prevent cation leaching (Johnson et al., 1982). The dominance of organic S as the major form of S is reasonably well established for all depths within the soil profile (David er al., 1983; Watwood et al., 1986, 1988; Autry et al., 1990). Autry et al. (1990) suggested that organic S formation may be as important as SO:- adsorption in retaining S at all depths within the soil profile for a variety of sites. The former process was shown to be predominantly bacterially mediated in the 02 and A horizons (Strickland and Fitzgerald, 1984). Fitzgerald (1973) has also documented the ability of cell-free extracts of Pseudomonas C,,B, a common soil isolate, to generate choline-o-sulfate, a form of organic S, from added SO:-, choline chloride, ATP, or MgCl,, implying that bacterial cells need to take up SO:- before incorporation of the anion into organic matter can occur. The influence of temperature (Strickland and Fitzgerald, 1984), soil pH (Watwood et al., 1986) and incubation time (Watwood et al., 1986, 1988) on the ability of soil to form organic S is well established. To date only cursory attention has been given to the effect that increasing SOi- concentration might have on organic S formation.

To predict how a given site will respond to SOiloading, it is necessary to determine the absolute maximum capacity that a given soil possesses to form organic S. This capacity should depend upon the kinetics of SO:- uptake by microorganisms within the soil. A versatile and powerful technique used to assess this capacity is the “heterotrophic activity method” described by Wright and Hobbie (1966). The method is a kinetic approach which describes the uptake kinetics for organisms when the natural substrate concentration is unknown which utilizes a linearization of the and Michaelis-Menton enzyme reaction which yields the equation: t/f = A /V,, + (Kt + S,)/ V,. . The term t is the incubation time, f the fraction of substrate taken up during time t, A is the concentration of added substrate, V,,,,, the maximal incorporation rate, Kt the half-saturation constant, and S, the endogenous substrate concentration (Wright and Hobbie. 1966). Kt can then be calculated if the latter concentration is known. If this concentration is not known, then (Kt + S,) can be taken as an upper limit on Kt (Azam and Hodson, 1981). The use of this technique has concentrated on assessing glucose mineralization activity in aquatic systems (Wright and Hobbie, 1966; Azam and Hodson, 1981; Ferroni et al., 1983) although Ferroni et al. (1985) have used it to measure glucose mineralization in soil ecosystems. The application of this technique to the study of SOi- uptake in soil has not hitherto been reported. The ability of a forest ecosystem to respond favorably to SOi- loading is related to the capacity of soil collected from the site to form organic S. When SO:- inputs exceed the amount required to bring the site to saturation with respect to organic S formation, any SOi- derived from higher atmospheric inputs is subject to leaching from the soil profile. Further study on determining the maximum capacity of a site to form organic S at all depths within the soil profile is thus warranted to better ascertain how a site will 143

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ANDREW

R. Au-ray and JOHNW. FITZCEMLD

respond to increasingly higher atmospheric inputs of SOi- for acidic deposition.

MATERIALS AND METHODS

Site description and sample collection Samples of the E (7-12 cm), Bs (17-26 cm), BC (40-50 cm) and C (50 + cm) horizons were collected from an excavated pit dug in a mixed, fir-spruce forest at the Howland site located near Orono, Maine. Soils from this site are in the Skerry series of moderately-drained Aquic Haplorthods. Root material and stones were removed by hand from samples, which were stored, field-moist, in sealed polyethylene bags at 4°C before analysis.

RESULTS

Figure la shows the effect of sodium axide on organic S formation in the Bs horizon. Even after the addition of azide, a baseline activity for this process was observed for all exposure times analyzed (Fig. la), and this is probably due to the activity of preformed enzymes present in the soil matrix. In the 1.2

(a)

Preparation of standard 35S-labelled surfate solutions A solution of 35S-labelled Na,SO, (ca 4 x 10” Bq mmol-‘ICN Pharmaceuticals) containing ca 1.67 x lo3 Bq ~1~’ was prepared. The final sulfate concentration was brought up to 2.0ymol ~1~’ by the addition of unlabelled anion. This solution was serially diluted, with the most dilute solution having a Sa- concentration of 3.75 x 10m3 nmol ~1~. All solutions were stored at 4°C before use. Time courses Triplicate samples (1 g wet wt, not sieved) were exposed at 20°C for varying times to either 7.5 nmol, 4Opmol or 200~1 of added Se,-. For those samples exposed to 7.5 nmol, half were incubated for the times indicated with 128mg g-t dry wt of sodium azide, a potent inhibitor of aerobic resiration. A salt extraction procedure (successive washes with 1 M Na*SO,, NaH*PO, and LiCl) was shown to quantitatively remove adsorbed 35S@,- and that subsequent extraction with 6 M HCl and 2 M NaOH removed 3% which had been incorporated into organic matter (Fitzgerald et al., 1982). Determination of organic S formation capacity Samples (1 g wet wt, not sieved) form each horizon were exposed in triplicate for 48 h at 20°C to 200 ~1 different standard solutions, with amounts of SOi- ranging from 20 pmol (1.67 x lo’ Bq) to 400 pmol (3.27 x lo5 Bq). Following exposure, samples were extracted according to the method of Fitzgerald et al. (1982) to yield soil water, salt, acid and base extracts, and the amount of organic S formed from a given concentration of added So’,was quantified as described for the time course studies. Statistical analysis Data derived from S incorporation studies was subjected to analysis of variance and Duncan’s Multiple Range Test (a = 0.05). Data derived from linearization was subjected to simple linear regression analysis.

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Fig. 1. Relationship between time and organic S formation by Bs horizon samples using: (a) 7.5 nmol added sulfate in the absenea (0) and presence of sodium azide (0); (b) differrnee in the amounts of organic S formed in the prr#na and absence of azide; (c) using 200 (W) and 40 (0) r mol added Se-.

Organosulfur formation in forest soils

745

absence of tide, a linear relationship between incubation *me up to 72 h &nd organic S formed from 7.5 nmol added SO:-’ was observed (Fig. la). When the organic S formation capacities for those samples incubated with azide were subtracted from those without azide, the capacity for organic S formation by living microbial cells can be determined, and the effect of exposure time on these capacities is demonstrated in Fig. I b. As was true for unamended samples, a linear relationship between exposure time and organic S formation capacity was observed (Fig. lb). In fact, the linearity of this process with respect to time was unaffected by the concentration of added SO:-, because for both 40 and 200 pmol of added SO:-, the process was linear with respect to time (Fig. lc). As was observed with 7.5 nmol of added SO:-, an initial burst of organic S was formed from the higher concentrations of SOi- (Fig. la, c). This was probably due to the baseline activity which failed to be abolished by the addition of azide. In light of these findings, we believe that this maximum capacity of a soil to incorporate SOi- into organic matter via microbial activity is a true potential, and hence we will use the term “saturation potential” to describe this capacity. The apparent rate of SOi- incorporation into organic matter, V_, derived from linear regression analysis of the Wright-Hobbie plots, generally decreased with increasing soil depth with the exception of the Bs horizon where a subs~ntia~ increase was observed (Table I). Saturation of organic S formation after 48 h was observed irrespective of depth, although these capacities, as well as the concentration of added So’,- required to bring the horizons to saturation varied considerably with increasing depth (Fig. 2, Table 1). A horizon is considered to be saturated if the mean amounts of organic S formed are not significantly different (ANOVA, Duncan’s Multiple Range Test, a = 0.05) over 25% of the concentration range examined. Linearization of organic S formation activities yielded straight lines for the E, Bs and C horizons (r* = 0.92, 0.89 and 0.98, respectively) and a curve for the BC horizon (Fig. 2 insert). If only the initial, linear portion of this curve is considered, however, a regression line can be drawn (r2 = 0.98), and one set of kinetic parameters for organic S formation, the system’s lower limit, can be calculated. Another way to determine the saturation potentials is to plot a regression line using the amounts of organic S on the saturation curve that are not significantly different. The saturation potential, derived in this manner, would then be numerically equal to the y-intercept of the fitted line (Fig. 2). For all horizons examined, the slope of this regression line was not significantly different from zero (a = 0.05), and values obtained for saturation potentials using this technique gave good agreement with those obtained by linearization for the E, BC and C horizons, but not for the Bs. Irrespective of the method used, the potential at saturation (the maximum capacity to incorporate sulfate into organic matter after 48 h) also followed this trend. Estimation of the SOi- concentration yielding saturation was assessed by two methods. The sum (Kt + S,), calculated as the x-intercept of the line in the Wright-Hobbie plots, gives a relative index of this

ANDREW

746

R. AUTRYand JOHNW. FITZGERALD

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Fig. 2. Relationship between increasing Se- concentration and organic S formation in samples collected from E, Bs, BC, and C horizons, respectively. 0, Relationship between organic S formed and w,- added; 0, Wright-Hobbie plot, where t,fand A are the incubation time, fraction of added S incorporated into organic matter, and concentration of added Sa-, respectively. capacity. As noted by Azam and Hodson (1981), if S,, the endogenous substrate concentration, is not

known, then (Kt + S,,) is an upper limit on Kt, the half-saturation constant. Doubling (Kt + S,) should then be an upper limit on the concentration of added Sa- yielding saturation. Another way to estimate this parameter is to determine whether the mean amounts of organic S formed are significantly different over 25% of the concentration range examined (ANOVA, Duncan’s Multiple Range Test, cc= 0.05). An estimate of the SOi- concentration yielding saturation can then be expressed as a range, with the lower limit being the concentration of added Seafter which all mean amounts of organic S formed from higher concentrations of added SO:- are not significantly different, and the upper limit being the next highest concentration of added Se,:- after the lower limit. A comparison of these two methods gave good agreement for all but the Bs horizon (Table I), where the value obtained for (Kt + S,) x 2 was out of the range determined by analysis of variance. While the saturation potentials followed a pattern with increasing depth, a similar statement could not be made for the Se,- concentration yielding saturation (Table 1). Turnover times (T,), the time required for complete mineralization of newly formed organic S, derived from Wright-Hobbie linearization, assuming a constant rate of removal and regeneration for

organic S, were calculated for all horizons, and also exhibited no obvious trend with increasing depth (Table 1). DISCUSSION

While a great deal is known about organic S formation capacities in litter and A horizon soils (Fitzgerald et al., 1982, 1988). little work has been done on assessing these capacities at greater depths within the soil profile. In the work that has been done, however, Swank et al. (1984) found that the organic S formation capacity of the Coweeta Hardwood site, using 7.5nmol of added SO:- decreased with increasing depth within the soil profile. A possible explanation for this observation is that decreases in microbial respiration and microbial biomass have been documented when comparing litter and uppermost mineral soil horizons (Cochran et al., 1989). Paul and Clark (1989) noted that microbial population size decreases throughout the rest of the soil profile. This suggests that rates of microbially-mediated processes, such as organic S formation, should decrease as a result of this decreased population size. Our findings for saturation potentials are similar in that these parameters also decreased with increasing depth, with the exception of the Bs horizon where an increase in this parameter

747

Organosulfur formation in forest soils

was observed. One possible explanation for this phenomenon is a higher bacterial population size in the Bs horizon. An increase in the bacterial population density was observed in B horizon samples taken from the Coweeta Hydrologic Laboratory (A. R. Autry and J. W. Fitzgerald, unpublished). This may be a function of bacterial adhesion to the soil of this horizon relative to others. Curvilinear relationships were observed with Wright-Hobbie plots of activity in the BC horizon. Such relationships were observed in glucose (Azam and Hodson, 1981) and phosphate Uarapchak and Herche, 1989) uptake studies using aquatic systems and indicate multiphasic uptake kinetics. This means that more than one microbial population was responsible for the uptake of the compound of interest (Azam and Hodson, 1981), whereas in the case of linear relationships in these plots, one or more microbial populations possessing indistinguishable kinetic parameters are responsible for SOi- uptake. The “differences” in microbial populations in the current study are based on these kinetic parameters, rather than physiological-phylogenetic species-specific differences. Hence, in the latter case, several phenotypically diverse populations could be responsible for SO,- uptake and yet not be distinguished from each other based solely on kinetic parameters for Sa- uptake. The kinetic data for the BC horizon, extrapolated from the linear portion of the Wright-Hobbie plot, closely matches that observed by inspection of the saturation curve. However, because the upper limit of the concentration range examined for these horizons was close to the crystallization point for So’- in solution, determination of this second set of parameters, which would include a higher V,, and Kt was considered meaningless and not attempted. The Wright-Hobbie approach (1966) yields another environmentally useful parameter, which is turnover time. The derivation and interpretation of this parameter are based on the assumption of steadystate conditions for the ecosystem. This assumption was made, but was not tested in this study. Values for this parameter, derived from linearization were in agreement with mobilization rates for recently formed organic S. In this latter work, Strickland et al. (1984) found that 68,61 and 26% of the organic S formed in the Al, Bl and B2 horizons, respectively, was mobilized within 24 h. Also, studies utilizing samples collected from sites in New Mexico by Watwood et al. (1986) show that incubation times > 24 h had no effect on mobilization rates for recently formed organic S. Therefore, turnover times of up to 907 h for total mineralization of recently formed organic S are not unreasonable. Only cursory attention has been given to the direct application of the Wright-Hobbie technique and its associated linearization to measuring soil processes. The bulk of the work has involved the use of nonlinear regression analysis to estimate kinetic parameters for mineralization or uptake of “C-labelled organic compounds by resident bacterial populations in the soil (Schmidt et al., 1985; Simkins and Alexander, 1985; Coody et al., 1986). More recently, Murray et al. (1989) used the Lineweaver-Burke linearization to measure nonlabelled NOi- utilization

by mixed populations of denitrifying bacteria in agricultural soils. Results obtained in our study indicate that the application of Wright and Hobbie’s heterotrophic activity method to SOi- uptake in soil ecosystems is valid. The linear Wright-Hobbie plot for the A horizon exhibited a high coefficient of determination (r2 > 0.96), and the curvilinear plot for the C horizon also exhibited a high coefficient of determination (r2 > 0.97). when only the linear portion of the curve was considered. Furthermore, comparison of the data obtained from linearization gave good agreement with that obtained by inspection of the actual saturation curves, in most cases. As noted previously (Wright and Hobbie, 1966; Williams, 1973; Azam and Hodson, 1981), application of the heterotrophic activity method to organic compound turnover time measurements is conditional, being valid only when A, the added substrate concentration, is close to S,, the endogenous substrate concentration. This is due to the fact that bacteria having low Kt values for uptake are major mineralizers of organic matter at low substrate concentrations, and their kinetic parameters are expressed in deference to other microbial populations under these conditions (Azam and Hodson, 1981). In our study, however, this condition was not observed, since interest was focused on assessing the maximal capacity of a given soil to form organic S. Another requirement for the application of this technique is that the exposure time be in a range where the incorporation rate is linear with respect to time (Ferroni et al., 1985). This was the case in our study. Additionally, Robinson and Tiedje (1983) noted that the use of Michaeli*Menton kinetics (and subsequent linearization) to describe microbial uptake of various compounds is valid only when this uptake is not coupled to microbial growth. Fitzgerald (1973) found that 0.1 mM concentrations of added Sa- to be sticient for growth of various Pseudomonas species, and also that these organisms release organic S into the culture medium. Obviously then, higher concentrations of added SO:-, such as those used in our study would likely not be growth-limiting, and hence uptake would not be linked to growth. Thus the conditions set forth by Robinson and Tiedje (1983) are satisfied, and the validity of the application of this method to SOi- incorporation studies in forest soil is confirmed. Acknowledgemenrs-This research was conducted as a part of the Integrated Forest Study and was funded by the Eleetrlc Power Research Institute. WC wish to thank I. Femandez for site selection and sampling. A.R.A. was supported by a university-wide assistantship awarded by the Graduate School of the University of Georgia. REFERENCES

Autry A. R., Fitzgerald J. W. and Caldwell P. R. (1990) Sulfur fractions and retention mechanisms in forest soils. Canadian JOWMI of Forest Research. 2Q, 337442.

Azam F. and Hodson R. (1981) Multiphasic kinetics for D glucose uptake by assemblages of natural marine bacteria. Marine Ecology Progress Series 6, 213-222. Cochran V. L., Elliott L. F. and Lewis C. E. (1989) Soil microbial biomass and enzyme activity in subarctic agricultural and forest soils. Biology and Fertility of Sails 7, 283-288.

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Cole D. W. and Johnson D. W. (1977) Atmospheric sulfate additions and cation leaching of a Douglas hr ecosystem. Water Resources Research 13, 313-317. Coody P. N., Sommcrs L. E. and Nelson D. W. (1986) Kinetics of glucose uptake by soil microorganisms. Soil Biology & Biochemistry 18, 283-289. David M. 9.. Schindler S. C.. Mitchell M. J. and Strick J. E. (1983) Importance of organic and inorganic sulfur to mineralization processes in a forest soil. Soil Biology & Biochemistry IS, 671677. Ferroni G. D., Leduc L. G. and Choquet C. G. (1983) Preliminary studies on the use of the heterotrophic activity method in simulated acid rain investigations of aquatic environments. Water Research 17, 1379-1384. Ferroni G. D., Leduc L. G. and Winterhalder E. K. (1985) The measurement of mineralization activity in soils. Soil Biology & Biochemistry 17, 727-728. Fitzgerald J. W. (1973) The formation of choline-o-sulphate by Pseudomonas C,sB and other Pseudomonas species. Biochemical Journal 136, 361-369. Fitzgerald J. W., Strickland T. C. and Swank W. T. (1982) Metabolic fate of inorganic sulphate in soil samples from undisturbed and managed forest ecosystems. Soil Biology & Biochemistrv 14. 529-536. Fitzgerald J. W*..,Swank W. T., Strickland T. C., Ash J. T., Hale D. D., Andrew T. L. and Watwood M. E. (1988) . , Sulfur .wols and transformations in litter and surface soil of a hardwood forest. In Ecological Studies: Forest Hydrology and Ecology at Coweeta, Vol. 66 (W. T. Swank and D. A. Crosslev Jr. Eds), pp. 246-253. Springer-Verlag, New York. _ __ Heute A. R. and McCall J. G. (1984) Soil cation leaching by ‘acid rain’ with varying nitrate to sulfate ratios. Journal of Environmental Quality 13, 366-371. Johnson D. W. (1980) Site susceptibility to leaching by HsSG, in acid rainfall. In E@cts of Acid Precipitation on Terrestrial Ecosystems (T. C. Hutchinson and M. Harms, Eds), pp. 525-535. Plenum Press, New York. Johnson D. W., Henderson G. S., Huff D. D., Lindberg S. E.. Richter D. D., Shriner D. S., Todd D. E. and Turner J. (1982) Cycling of organic and inorganic sulphur in a chestnut oak forest. Oecoloaia 54, 141-148. Likens G. E. and Bormann F. H. (1974) Acid rain: a serious regional environmental problem. Science 184, 1176-1179. Murray R. E., Parsons L. L. and Smith M. S. (1989) Kinetics of nitrate utilization by mixed populations of

bacteria. Applied and Environmental Microbiology 55, 717-721. Paul E. A. and Clark F. E. (1989) Occurrence and distribution of soil organisms. In Soil Microbiology and Biochemistry, pp. 74-90. Academic Press, San Diego. Robinson J. A. and Tiedje J. M. (1983) Nonlinear estimation of Monod growth kinetic parameters from a single substrate depletion curve. Appkd and Environmental Microbiology 45, 1453-1458. Schmidt S. K., Simkins S. and Alexander M. (1985) Models for the kinetics of biodegradation of organic compounds not supporting growth. Applied and Environmental Microbiology 50, 323-33 1. Simkins S. and Alexander M. (1985) Nonlinear estimation of the parameters of Monod kinetics that best describe mineralization of several substrate concentrations by dissimilar bacterial densities. Applied and Environmental Microbiology 50, 816-824. Strickland T. C. and Fitzgerald J. W. (1984) Formation and mineralization of organic sulfur in forest soils. Biogeochemistry 1, 79-95. Strickland T. C., Fitzgerald J. W. and Swank W. T. (1984) Mobilization of recently formed forest soil organic sulfur. Canadian Journal of Forest Research 14; 63-67. Swank W. T., Fitzgerald J. W. and Ash J. T. (1984) Microbial transformations of sulfate in forest soils. Science 223, 182-184. Tarapchak S. J. and Herche L. R. (1989) Phosphate uptake by microbial assemblages: model requirements and evaluation of experimental methods. Journal of Environmental Quality 18, 17-25. Watwood M. E., Fitzgerald J. W. and Gosx J. R. (1986) Sulfur processing in forest soil and litter along an elevational and vegetative gradient. Canadian Journalof Forest Research 16, 689-695. Watwood M. E., Fitzgerald J. W., Swank W. T. and Blood E. R. (1988) Factors involved in potential sulfur accumulation in litter and soil from a coastal pine forest. Biogeocbemistry 6, 3-19. Williams P. J. L. (1973) The validity of the application of simple kinetic analysis to heterogenous microbial pop&ions. Limnology and Oceanography 18, 159-165. Wright R. T. and Hobbie J. E. (1966) Use of glucose and acetate by bacteria and algae in aquatic ecosystems. Ecology 47, 447-464.