Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1982, p /82/ $02.00/0 Vol. 43, No. 6 Fluorescein Diacetate Hydrolysis as a Measure of Total Microbial Activity in Soil and Litter JOHAN SCHNURER* AND THOMAS ROSSWALL Department of Microbiology, Swedish University ofagricultural Sciences, S Uppsala, Sweden Received 3 December 1981/Accepted 13 March 1982 Spectrophotometric determination of the hydrolysis of fluorescein diacetate (FDA) was shown to be a simple, sensitive, and rapid method for determining microbial activity in soil and litter. FDA hydrolysis was studied in soil and straw incubated for up to 3 h. Hydrolysis was found to increase linearly with soil addition. FDA hydrolysis by pure cultures of Fusarium culmorum increased linearly with mycelium addition both in shake cultures and after inoculation into sterile soil. FDA hydrolysis by Pseudomonas denitrificans increased linearly with biomass addition. The FDA hydrolytic activities in soil samples from different layers of an agricultural soil were correlated with respiration. Acetone was found to be suitable for terminating the reaction. Total microbial activity is a good general measure of organic matter turnover in natural habitats since generally more than 90% of the energy flow passes through microbial decomposers (6). Many criteria for determining overall microbial activity in soil, litter, and water have been used: for example, respiration (16), dehydrogenase activity (2, 13), and heat evolution determined by microcalorimetry (11). A technique suitable for measuring total microbial activity must be nonspecific and sensitive, and any incubation period necessary should be as short as possible. Fluorescein diacetate (3',6'-diacetylfluorescein [FDA]) has been used to determine amounts of active fungi (14) and bacteria (1, 8) and to locate acetylesterases in living protist cells (10). FDA is hydrolyzed by a number of different enzymes, such as proteases, lipases, and esterases (5, 12). The product of this enzymatic conversion is fluorescein, which can be visualized within cells by fluorescence microscopy. Fluorescein can also be quantified by fluorometry or spectrophotometry. Swisher and Carroll (15) have used this technique to determine the amount of active microbial biomass in needle litter by incubating needles with an FDA solution and determining the resulting amount of fluorescein spectrophotometrically. This paper reports the use of FDA to determine total microbial activity in soil and straw litter. The FDA hydrolytic activity was also compared with respiration rates of samples from different depths of an agricultural soil. MATERIALS AND METHODS Soil. The soil was collected from an agricultural field at Kjettslinge (Uppland, Sweden). The soil consists of 1256 three distinct layers: the topsoil, a sandy loam (27 cm thick [P.-E. Jansson, personal communication]; 6.6% loss on ignition; 0.25% N; ph 6.7); a sand layer (0.5% loss on ignition; 0.02% N; ph 7.0) with a mean thickness of 15 cm (Jansson, personal communication); and a clay layer (3.0% loss on ignition; 0.04% N; ph 7.3). For most experiments, the topsoil was sifted through a sieve (2-mm mesh) and stored at 5 C before being used. Freshly collected soil was used in the experiments with soil samples from different depths. We sterilized soil samples by autoclaving them at 120 C for 20 min on 2 consecutive days. Straw. Barley straw (0.6% N) was harvested in the autumn, cut in 2-cm pieces, and stored at room temperature. We sterilized the straw by autoclaving it at 110 C for 30 min. Microorganisms. Pseudomonas denitrificans (NCIB 8376) and Fusarium culmorum ((W.G. Sm.) Sacc.) were used for pure-culture studies of FDA hydrolysis. P. denitrificans was grown in nutrient broth (Oxoid Ltd., London) (concentration of broth in water, 13 g/ liter) on a rotary shaker for 24 h at 28 C before the experiments were started. To prevent the culture broth from influencing the hydrolysis of FDA, we harvested the cells by centrifugation at 6,000 rpm for 5 min and washed them twice with 60 mm sodium phosphate buffer, ph 7.6. F. culmorum was selected because it is a common soil inhabitant with good competitive saprophytic ability (4). The fungus was grown on malt extract broth (Oxoid) (concentration of broth in water, 20 g/liter) at 24 C for 5 days or until heavy sporulation could be observed. We filtered the culture through glass wool to obtain a filtrate of smaller propagules. A few drops of this filtrate were used to inoculate the 150 ml offresh malt extract broth in each 500-ml Erlenmeyer flask, which also contained 20 small glass beads. This procedure was necessary to obtain approximately synchronous germination and mycelial growth without pellet formation. The cultures were grown on a rotary shaker for 48 h at 24 C before the experiments were started. To prevent the culture broth from influencing the hydrolysis of FDA, we

2 VOL. 43, 1982 FDA HYDROLYSIS IN SOIL AND LITTER 1257 am / /---~~ -l Incubation time (min) FIG. 1. Hydrolysis of FDA by straw samples over time (1.0 g [dry weight] of straw in 100 ml of 60 mm sodium phosphate buffer, ph 7.6). Means of three replicate determinations are shown. Standard deviation, <5%. Symbols: 0, nonautoclaved straw; 0, autoclaved straw. harvested the mycelium by centrifugation at 4,000 rpm for 5 min and washed it twice with 60 mm sodium phosphate buffer, ph 7.6. We determined the dry weights of P. denitrificans and F. culmorum suspensions by drying them for 20 h at 85 C. Determination of FDA hydrolysis. FDA (Sigma Chemical Co., St. Louis, Mo.) was dissolved in acetone (analysis grade; E. Merck AG, Darmstadt, Germany) and stored as a stock solution (2 mg/ml) at -20 C. The amount of FDA hydrolyzed was measured as absorbance at 490 nm (A490) with a Beckman model 24 spectrophotometer equipped with a Beckman Clinical Sippersystem, which permitted the rapid handling of many samples. Samples giving absorption values of greater than 0.8 were always diluted before final absorbance determinations. For all determinations of FDA hydrolytic activity, the sample (soil, straw, bacterial or fungal suspension) was simultaneously added with FDA (final concentration, 10,ug/ml) to sterile 60 mm sodium phosphate buffer, ph 7.6, and the mixture was incubated at 24 C on a rotary shaker. The buffering capacity was sufficient to keep the ph at 7.6 for the duration of the experiments. In the experiments with straw, A490 values could be determined directly in buffer samples taken from the incubation flasks. P. denitrificans cells were removed from the incubation solution by centrifugation for 5 min at 6,000 rpm, and the supernatant was filtered 0 through Munktell no. 3 filter paper (Stora Kopparberg AB, Grycksbo, Sweden). In studies of F. culmorum, the mycelium was removed by centrifugation at 6,000 rpm for 5 min followed by filtration of the supernatant through a 3-,um membrane filter (Millipore Corp., Bedford, Mass.). Soil was removed from the incubation solution by centrifugation for 5 min at 6,000 rpm followed by filtration through Munktell no. 3 filter paper. This produced a clear solution with a low background absorbance (mostly below 0.05). In some experiments, the hydrolysis of FDA was terminated by the addition of acetone (final concentration, 50o [vol/vol]). This had the additional effect of reducing the background absorbance of the clay samples to below Without the addition of acetone, it was sometimes difficult to remove colloids from the supernatants of clay samples. We determined the adsorption of hydrolyzed FDA to soil by adding soil to solutions of prehydrolyzed FDA with a known A490 value (-0.5). We hydrolyzed FDA by placing a flask with FDA in phosphate buffer (10,ug/ml) in a boiling water bath for 30 min. The soil solutions were incubated on a rotary shaker at 24 C for 60 or 120 min. A490 was determined after removal of the soil by centrifugation and filtration. Respiration was determined as oxygen consumption in a Gilson respirometer at 15 C. Respiration readings were taken at approximately half-hour intervals for at least 3 h. RESULTS Straw. The FDA hydrolytic activity of straw increased linearly with time during the first 2 h (Fig. 1) and then slowed down. The hydrolytic activity of autoclaved straw was low compared with that of nonautoclaved straw. The spontaneous hydrolysis of FDA was found to be low (<0.03 A490 in 3 h). Soil. The hydrolysis of FDA increased approximately linearly with time (Fig. 2) and amount of soil (Fig. 3). Autoclaved soil did not show any FDA hydrolytic activity. Pure cultures of F. culmorum and P. denitrificans. The hydrolysis of FDA by different amounts of fungi and bacteria was approximately linear until A490 = 1.2 (Fig. 4). F. culmorum in autoclaved soil. The hydrolysis of FDA was found to increase with the addition of fungi to autoclaved soil (Fig. 5). Termination of FDA hydrolysis with acetone. Because of the rapidity of FDA hydrolysis, it was necessary, when working with many samples, to find a way of terminating hydrolysis at a specific time. Since FDA is heat and ph labile (5), this proved difficult. Attempts to terminate hydrolysis with different neutral salts, metal salts, and various organic solvents were made. Acetone (50% [vol/vol]) was found to be the most efficient: it totally stopped hydrolysis in a soil sample for 2 h (Fig. 6). FDA hydrolysis and respiration of field samples. A good correlation was found between FDA hydrolytic activity and 02 consumption in

3 1258 SCHNURER AND ROSSWALL APPL. ENVIRON. MICROBIOL.. o 0.25 /1 // Incubation time (min) FIG. 2. Hydrolysis of FDA by soil samples over time (1.7 g [dry weight] of soil in 100 ml of 60 mm sodium phosphate buffer, ph 7.6). Means of three replicate determinations are shown. Bars indicate standard deviations. soil samples collected from different depths of the agricultural soil tested (Table 1). Most of the activity was found in the topsoil, whereas very limited activity was found in the lower layers. Using the same soil amounts and time intervals used for the field measurements, we found that the adsorption of fluorescein to soil did not exceed 7% and was mostly lower than 5% i I I I Amount of soil (g d.w.) FIG. 3. Hydrolysis of FDA by different amounts (per 50 ml of 60 mm sodium phosphate buffer, ph 7.6) of soil incubated for 30 min. Means of three replicate determinations are shown. Standard deviation, <5%. Q Fungal or bacterial biomass (mg d.w.) FIG. 4. Hydrolysis of FDA by different amounts of F. culmorum (13 mg [dry weight]/ml) or P. denitrificans (10 mg [dry weight]/ml) suspensions. The suspensions were placed in test tubes, and 60 mm sodium phosphate buffer, ph 7.6, was added to give a final volume of 10 ml in each tube. The test tubes were incubated with FDA on a rotary shaker for 60 min at 24 C. Means of three replicate determinations are shown. Standard deviation, <5%. Symbols: *, F. culmorum; x, P. denitrificans. DISCUSSION FDA as a substrate for determining the overall activity of decomposer microorganisms seems promising, judged by the results obtained for straw litter (Fig. 1) and soil (Fig. 2 and 3). The method was used by Swisher and Carroll (15) for determining microbial biomass on coniferous needles, and these researchers indicated that the method could be used for determining the microbial biomass in a variety of habitats. It is probably more rewarding to use FDA as a substrate for determining heterotrophic activity than to use it for determining biomass. In the present investigation, no attempt was made to relate the observed total FDA activity to the active microbial biomass determined by FDA microscopic techniques (8, 14). The active biomass of a heterogeneous microbial population metabolizing FDA is probably not directly proportional to the total FDA hydrolysis as measured by the technique described above, and FDA microscopy only distinguishes between active and inactive biomasses and does not indicate the level of activity of the active bio-

4 VOL. 43, 1982 FDA HYDROLYSIS IN SOIL AND LITTER I I I I Amount of fungi (mg d.w. * gl d.w. soi l) FIG. 5. Hydrolysis of FDA by different amounts of F. culmorum in autoclaved soil. Different amounts of fungal mycelium were mixed with 60 mm sodium phosphate buffer, ph 7.6, to give a final volume of 5 ml. This volume was mixed with 22.0 g (dry weight) of autoclaved soil. Soil (2.2 g) was then transferred to 50 ml of buffer and incubated with FDA for 180 min. Means of three replicate determinations are shown. Bars indicate standard deviations. mass. The aim of this paper was not to determine total active biomass but to suggest a simple and sensitive method for determining total microbial activity. The difficulties of relating mi I Incubation time (min) FIG. 6. Termination of FDA hydrolysis with acetone. Soil (4 g [dry weight]) in 100 ml of 60 mm sodium phosphate buffer was incubated with FDA on a rotary shaker at 24 C. After 62 min (arrow), phosphate buffer or acetone was added to a 50% (vol/vol) final concentration. Means of six (first 60 min) and three replicate determinations are shown. Standard deviation, <5%. Symbols: 0, undiluted; x, 50% phosphate buffer added; 0, 50%o acetone added. TABLE 1. FDA hydrolytic activity and respiration (at 15 C) of samples from different depths of an agricultural soila FDA hydrolytic Respiration (ILl of Soil Soil layer activity (A490 units/h and g 02/h and g [dry wtl) [dry wtj) Topsoil ( ± cm) Topsoil (10 cm 0.19 ± ± 0.11 to sand layer) Sand layer 0.02 ± Clay layer (up ± ± 0.06 per 10 cm) a Samples (2 g [wet weight]) were incubated with 50 ml of buffer (as described in the text) for the determination of FDA hydrolysis. Topsoil samples were incubated for 60 min, and samples from the sand and clay layers were incubated for 120 min. Hydrolysis was terminated with 50% (vol/vol) acetone. Respiration was determined for 40- to 80-g (wet weight) samples. Values are means for four samples ± standard deviations. crobial activity to microbial biomass determined by, for example, enzyme assays or ATP determinations are well known (3). Earlier studies have shown that all fungi investigated (10, 14), most bacteria (8, 10), and some protozoa and algae (10) exhibit FDA hydrolytic activity. FDA has also been used in studies of the enzymatic activities of mammalian cells (12). The ability to hydrolyze FDA thus seems widespread, especially among the major decomposers-bacteria and fungi. The decrease in the rate of FDA hydrolysis at higher amounts of fungal biomass in liquid culture (Fig. 4) was probably due to substrate limitation. This could also explain the decline in the rate of hydrolysis observed in straw (Fig. 1). It is not possible to increase substrate concentration without reducing result reproducibility, owing to a slightly cloudy FDA solution at higher substrate concentrations (5). There was an indication of a slight lag phase in FDA hydrolytic activity in straw (Fig. 1); this lag phase was more prominent in some of the experiments with soil (Fig. 6). This is probably due to a lag phase occurring before maximal FDA uptake by individual cells and also to the fact that fluorescein is liberated into the environment only after the storage capacity of the cells has been exceeded (18). FDA has been shown to be nontoxic for bacteria (10) and mammalian cells (12) at the concentration used in the present investigation. The low activity observed when F. culmorum was added to soil (Fig. 5), compared with that observed in the pure culture (Fig. 4), was due to the difference in fungal biomass sizes in the two

5 1260 SCHNURER AND ROSSWALL experiments: 0.22 mg (dry weight)/ml for F. culmorum added to soil and 6.5 mg/ml for the pure culture. A good correlation was found between FDA hydrolysis and respiration (Table 1), and the differences among the soil layers in decomposer activity was probably a reflection of the lower amounts of organic soil matter at greater depths. Similar correlations among different measures of microbial activity have been observed by other authors (3, 17). The FDA activity was very low in the sand and clay samples, and the amounts of soil used should have been increased so that an incubation time of no more than 1 h could have been used. Determination of FDA hydrolysis is only one of a number of methods available for the assessment of microbial activity in samples from natural habitats. AU methods have their limitations, and the selection of one for a particular investigation is determined by a number of factors. ATP analysis has one significant advantage over all other methods: no incubation is needed since the analysis is made on ATP extracted from cells. The method is very sensitive but requires careful extraction to prevent the hydrolysis of the ATP (7). Special equipment is also needed. Respiration measurements have the advantage of directly measuring CO2 (or 02) and thus providing a quantitative measurement of carbon flux through the investigated system. Respiration determinations generally require an incubation time of several hours (16) and, compared with enzyme assay techniques, are more difficult in terms of the handling of large numbers of samples. The determination of dehydrogenase activity has also been a common measurement of activity, but this assay is not very sensitive and requires many hours of incubation if the microbial activity is low (13). The determination of FDA hydrolysis has the advantage of being simple, rapid, and sensitive, and it should prove useful, especially for comparative studies of microbial activity in natural habitats. Acetone was used to terminate the reaction only in the last two experiments. The rapidity of the FDA hydrolysis makes it necessary to determine absorbance at accurately determined times if the reaction cannot be terminated by the use of an efficient inhibitor. To facilitate the assay of large numbers of samples, the use of acetone is suggested: it inhibited the enzymatic reaction rapidly and completely. In preliminary experiments, it was shown that an acetone concentration of at least 40% was needed to terminate the reaction. When acetone was added, there was a greater decrease in the absorbance of an FDA solution than that observed when buffer was added (Fig. 6), but preliminary experiments with APPL. ENVIRON. MICROBIOL. heat-hydrolyzed FDA (A490, 0.25 to 1.5) showed that the percent decrease in A490 was independent of initial fluorescein concentration. Moreover, acetone helped solubilize cell membranes, facilitating the extraction of the fluorescein formed. Because of its polarity, fluorescein is poorly transported through the cell membranes (12), and only the excess is excreted (18). Problenis may be encountered with the FDA method at high and low phs, at which nonbiological hydrolysis of FDA may occur. The adsorption of fluorescein to soil particles should be checked for, but this does not seem to be a real problem, even in soils with high clay contents. A need for screening a large number of samples for microbial activity often arises, and we believe that the determination of FDA hydrolysis as a general indicator of microbial activity shows great promise. ACKNOWLEDGMENTS We are grateful to Inger Ohlsson for skillful technical assistance. The project was supported by grants from the Swedish Council for Planning and Coordination of Research, the Swedish Council for Forestry and Agricultural Research, the Swedish Natural Science Research Council, and the Swedish Environment Protection Board to the project "Ecology of Arable Land. Role of Organisms in Nitrogen Cycling." LITERATURE CITED 1. Brunius, G Technical aspects of the use of 3',6'- diacetyl fluorescein for vital fluorescent staining of bacteria. Curr. Microbiol. 4: Casida, L. E., Jr., D. A. Klein, and T. Santoro Soil dehydrogenase activity. Soil Sci. 98: Domsch, K. H., T. Beck, J. P. E. Anderson, B. Soderstrom, D. Parkinson, and G. Trolldenier A comparison of methods for soil microbial population and biomass studies. Z. Pflanzenernaehr. Bodenkd. 142: Garret, S. D Soil fungi and soil fertility. Pergamon Press, Oxford, England. 5. Guilbault, G. G., and D. N. Kramer Fluorometric determination of lipase, acylase, alpha- and gamma-chymotrypsin and inhibitors of these enzymes. Anal. Chem. 36: Heal, 0. W., and S. F. MacLean, Jr Comparative productivity in ecosystems-secondary productivity. In W. H. van Dobben and R. H. Lowe-McConnell (ed.), Unifying concepts in ecology, p Dr. W. Junk B. V. Publishers, The Hague, Holland. 7. Jenkinson, D. S., and J. M. Oades A method for measuring adenosine triphosphate in soil. Soil Biol. Biochem. 11: Lundgren, B Fluorescein diacetate as a stain of metabolically active bacteria in soil. Oikos 36: MacLeod, N. H., E. W. Chapelle, and A. M. Crawford ATP assay of terrestrial soils; a test of an exobiological experiment. Nature (London) 223: Medzon, E. L., and M. L. Brady Direct measurement of acetylesterase in living protist cells. J. Bacteriol. 97: Mortensen, U., B. Norin, and I. Wadso Microcalorimetry in the study of the activity of microorganisms. Bull. Ecol. Res. Comm. (NFR Statens Naturvetensk. Forskningsrad) 17: Rotman, B., and B. W. Papermaster Membrane properties of living mammalian cells as studied by enzy-

6 VOL. 43, 1982 FDA HYDROLYSIS IN SOIL AND LITTER 1261 matic hydrolysis of fluorogenic esters. Proc. Nati. Acad. Sci. U.S.A. 55: Smith, S. N., and G. J. F. Pugh Evaluation of dehydrogenase as a suitable indicator of microbial activity. Enzyme Microb. Technol. 1: S6derstr6m, B. E Vital staining of fungi in pure cultures and in soil with fluorescein diacetate. Soil Biol. Biochem. 9: Swisher, R., and G. C. Carroll Fluorescein diacetate hydrolysis as an estimator of microbial biomass on coniferous needle surfaces. Microb. Ecol. 6: Van Cleve, K., P. I. Coyne, E. Goodwin, C. Johnson, and M. Kelley A comparison of four methods for measuring respiration in organic material. Soil Biol. Biochem. 11: Witkamp, M Compatibility of microbial measurements. Bull. Ecol. Res. Comm. (NFR Statens Naturvetensk. Forskningsrad) 17: Ziegler, G. B., E. Ziegler, and R. Witzenhausen Nachweis der Stoffwechselaktivitet von Mikroorganismen durch Vital-Fluorochromierung mit 3',6'-Diacetylfluorescein Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 230: (In German with English summary.)