Vol. 43, No. 6 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1982, p. 1256-1261 0099-2240/82/061256-06$02.00/0 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-750 07 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 decom- posers (6). Many criteria for determining overall microbial activity in soil, litter, and water have been used: for example, respiration (16), dehy- drogenase activity (2, 13), and heat evolution determined by microcalorimetry (11). A tech- nique suitable for measuring total microbial ac- tivity must be nonspecific and sensitive, and any incubation period necessary should be as short as possible. Fluorescein diacetate (3',6'-diacetylfluores- cein [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 enzy- matic conversion is fluorescein, which can be visualized within cells by fluorescence micros- copy. Fluorescein can also be quantified by fluorometry or spectrophotometry. Swisher and Carroll (15) have used this technique to deter- mine 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 micro- bial activity in soil and straw litter. The FDA hydrolytic activity was also compared with res- piration 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 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 communica- tion) 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 1256

FDA HYDROLYSIS IN SOIL AND LITTER 2.0 1.5 am 1.01 0.51 0 0 / /---~~ -l 60 120 180 240 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 devi- ation, 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 suspen- sions by drying them for 20 h at 85��C. Determination of FDA hydrolysis. FDA (Sigma Chemical Co., St. Louis, Mo.) was dissolved in ace- tone (analysis grade E. Merck AG, Darmstadt, Ger- many) 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 Clini- cal 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 concentra- tion, 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 suffi- cient 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 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 incuba- tion 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 concentra- tion, 50o [vol/vol]). This had the additional effect of reducing the background absorbance of the clay sam- ples to below 0.02. 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 spontane- ous hydrolysis of FDA was found to be low (0.03 A490 in 3 h). Soil. The hydrolysis of FDA increased ap- proximately 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. denitrifi- cans. The hydrolysis of FDA by different amounts of fungi and bacteria was approximate- ly 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 sam- ples, 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 sam- ples. A good correlation was found between FDA hydrolytic activity and 02 consumption in VOL. 43, 1982 1257