Environ. Sci. Technol. 2004, 38, 5134-5140
Azo Dye Method for Mapping Relative Sediment Enzyme Activity in Situ at Precise Spatial Locations NICOLA J. ROGERS* AND SIMON C. APTE Centre for Environmental Contaminants Research, CSIRO Energy Technology, Private Mail Bag 7, Bangor, New South Wales 2234, Australia
Existing methodology for measuring microbial enzyme activity in aquatic sediments involves horizontal sectioning of sediment cores into centimeter slices, followed by determination of the enzyme activity of each homogenized sediment slice. At best, this approach provides only onedimensional information on the distribution of microbial activity. This paper describes the development of a novel technique to map sediment enzyme activity in situ at millimeter spatial resolution. Naphthol AS enzyme substrates were loaded onto filter membranes by evaporation from an organic solvent. The membranes were attached to plastic cards to form rigid probes, which were deployed vertically in sediments for a fixed time period. The exposed membranes were developed in a diazonium salt solution, resulting in the formation of a colored precipitate where substrate hydrolysis had occurred. The chromogenic reaction was calibrated and quantified by immersing substrateloaded membranes in a series of solutions of known enzyme activity. A flatbed scanner and image analysis software were used to produce digitized images and to generate two-dimensional maps of enzyme activity. The technique was used to map the spatial features of esterase activity in aquatic sediment samples from wetland areas and enabled the precise locations of microbial activity “hotspots” to be identified.
Introduction Traditional models often describe sediment processes as one-dimensional systems with vertical structure. This view has arisen from concentration-depth analyses of solid and pore water components measured at centimeter resolution. However, recent developments in the fine-scale geochemical characterization of sediments do not support such a simple model (1). Microelectrodes (2-4) and optical microsensors (5) have been used for many years to make measurements of oxygen distribution and of nutrient ion fluxes in aquatic systems. More recently, planar optical electrodes (6, 7) have been developed to measure the two-dimensional fine structure of oxygen distributions in benthic microbial communities. Diffusive gel probes have been used to map highly localized zones of trace metal remobilization at millimeter (8), submillimeter (9), and two-dimensional (10) resolution in sediment pore waters. Nutrient accumulation has similarly been measured using diffusive gel methods (11, 12). These studies have highlighted the horizontal and vertical heterogeneity of sediment chemistry at high spatial (sub-mm) resolution. * Corresponding author phone: 61-2-9710-6853; fax: 61-2-97106837, e-mail:
[email protected]. 5134 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 19, 2004
The microscale features of aquatic sediments have previously been described in terms of sediment structure (13) and the distribution of microbial communities on sediment grains (14, 15), and the occurrence of small, highly localized zones of microbial activity has often been suggested (1, 16). As far as we are aware however, few attempts have previously been made to make in situ fine-scale spatial measurements of the distribution of sediment enzyme activity. Existing methods for measuring microbial activity in sediments involve horizontal sectioning of sediment cores into cm slices, followed by determination of enzyme activity using a labeled substrate, which is added to slurries of each homogenized sediment slice (17). At best this approach provides only relatively crude information on the distribution of enzyme activity in sediments, giving a one-dimensional vertical profile at centimeter resolution. Higher resolution measurements of microbial enzyme activity under in situ conditions may contribute to an increased understanding of the interactions between biological and geochemical processes. An in situ sensor or probe to measure microbial enzyme activity in sediments has several desirable criteria. The use of simple, commercially available, methodology such as colorimetry or fluorimetry and a straightforward detection system would allow the probes to be easily prepared and analyzed. The substrate and reaction products must be immobilized on the probe, so that neither the reactants nor the products can diffuse away into the surrounding aqueous environment. Spatial resolution of enzyme activity at the millimeter (or even submillimeter) scale is required and, ideally, the technique should be compatible with currently available in situ techniques used for characterizing geochemical processes in sediments. Azo dyes have been used for the histochemical detection and localization of enzyme activity in tissue sections for many decades (18). These techniques involve the application of an essentially insoluble chromophore (usually a naphthol) with an attached substrate group, to a fixed tissue section, in the presence of a diazonium salt. Enzymes in the tissues hydrolyze the substituted naphthol, which is subsequently coupled to the diazonium salt resulting in the formation of a brightly colored precipitate at the site of enzymatic activity. Excellent localization of areas of enzyme activity is achieved and visualized under microscopic examination. Naphthol AS enzyme substrates were first described by Burstone (19) and are now widely available with a variety of functional group substitutions. They are reported to have an aqueous solubility of less than 1 µg mL-1, far lower than any other substituted naphthols (18, 19). Many enzyme activities have been detected in sediments and there is potential to investigate several enzymes from the major metabolic pathways (C, P, N, S) when assessing overall microbial activity. Microbial ectoenzymes exhibiting esterase activities are involved in many different processes in freshwaters. Chro´st and co-workers (20, 21) showed that the activity of microbial esterases in lake water was significantly positively correlated to activities of aminopeptidase β-glucosidase, β-galactosidase, and lipase, and suggest that esterase activity might be a useful indicator of the overall potential activity of microbial enzymes in the biogeochemical transformations of organic matter. Although the insolubility of various naphthol substrates has been considered a hindrance in histochemical studies (18), this has been exploited to positive benefit in the current work. Histochemical methods have been adapted to develop a novel sensor to measure esterase activity in sediments in situ and under ambient conditions. Naphthol AS enzyme 10.1021/es049891r CCC: $27.50
Published 2004 by the Am. Chem. Soc. Published on Web 08/24/2004
substrates were dissolved in an organic solvent and the resulting solution was applied to a filter membrane. The solvent was then allowed to evaporate, generating an even coating of an essentially insoluble enzyme substrate on the surface of the filter membrane. The enzyme substrate and all subsequent reaction products are insoluble in aqueous solution and thus remain localized on the sensor throughout the process. A two-step process was required for the detection of enzyme activity. The substrate-loaded filter membranes were exposed to enzyme solutions, or aquatic sediments, for up to 24 h and then removed and rinsed with distilled water. Following exposure, the membranes were “developed” in a diazonium salt solution resulting in the formation of a colored precipitate where substrate hydrolysis had occurred. A flat bed scanner and computer-imaging densitometry (12) were used to analyze the colorimetric data generated by the sensor and to produce spatial maps of enzyme activity and microbial distribution.
Methods Reagents. Cotton cellulose filter papers, Quantitative No. 6, were obtained from Advantec, Toyo Kaisha, Ltd (Tokyo, Japan). Polysulfone membrane filter papers with a 45-µm pore size, Supor 450 and HT Tuffryn 450, were obtained from Pall Life Sciences (Ann Arbor, MI). The enzyme substrates naphthol AS-MX acetate, naphthol AS-D acetate, naphthol AS-MX butyrate, and naphthol AS-MX phosphate, commercial preparations of the hydrolysis products Naphthol AS-MX (3-hydroxy-2-naphthoic acid 2,4-dimethylanilide) and naphthol AS-D (3-hydroxy-2-naphthoic acid o-toluidide), and the diazonium salt Fast Red TR (hemi [zinc chloride] 4-chloro2-methylbenzenediazonium salt) were obtained from SigmaAldrich, Inc. (St Louis, MO). A working solution of esterase extracted from porcine liver (Sigma-Aldrich) was prepared by dissolving the solid (41 units mg-1 solid) in deionized water to achieve a final concentration of 2 units mL-1. The working solution of esterase was kept refrigerated (4 °C) and used for up to fourteen days. A stock solution of alkaline phosphatase from Escherichia coli (Worthington Biochemical Corporation, Lakewood, NJ) was prepared by dissolving 10 mg (193 units) in 100 mL of deionized water to give a final working activity of approximately 2 units mL-1 and kept refrigerated at 4 °C. Filter Preparation. Cellulose or polysulfone membrane filters were handled as little as possible prior to application of the enzyme substrate to minimize damage and subsequent uneven substrate coverage. A histochemical enzyme substrate, e.g., naphthol AS-MX acetate, was dissolved in acetone or methanol, typically 10 mg mL-1 (0.03 M), to produce a colorless solution. A filter membrane, typically 6 cm by 8 cm (48 cm2), was placed on a clean, dry, glass surface and a 1 mL aliquot of the enzyme substrate solution was applied to the membrane using a micropipet ensuring that the entire surface of the filter was wetted with the solution. The solvent was allowed to evaporate at room temperature until the membrane was completely dry, achieving a final substrate loading of approximately 0.6 µmol cm-2. A treated filter or membrane was attached to the face of a thin plastic card (5.5 cm by 8.5 cm by 30 mM). Selection of the Filter Support. Three types of filter membrane, HT-Tuffryn and Supor 450 polysulfone membranes and cotton-cellulose filters, were evaluated. Initial experiments indicated that an uneven loading of the enzyme substrate was obtained on the HT-Tuffryn membrane with all the substrates tested. The use of Supor 450 improved the uniformity of loading. This membrane had better wetting properties when treated with the naphthol-derived enzyme substrates and reproducible results were obtained. This was possibly a reflection of a higher flow rate and slightly reduced thickness compared to the HT-Tuffryn 450. However, insufficient enzyme substrate could be loaded onto the Supor 450 membrane and the technique became substrate limiting when the probes were deployed in sediments for extended time periods (24 h). A uniform, low intensity, color was observed over all areas of the membrane. Polysulfone filter membrane was not chemically resistant to acetone so 5136
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 19, 2004
substrate loadings higher than 0.2 µmol cm-2 could not be investigated. Subsequently cotton-cellulose filters were used as the support for the enzyme substrate. These filters produced excellent uniformity of application, reproducibility of results, and allowed sufficient loading of the substrate to differentiate between areas of high and low enzyme activity in aquatic sediments. Optimization of Substrate Loading. Increasing concentrations of the enzyme substrate, naphthol AS-MX acetate, were applied to 4.8 cm2 cellulose membrane filters, which were exposed to an excess enzyme concentration (2 units mL-1, 10 mL) for 24 h prior to development in Fast Red TR. The maximal loading on cellulose occurred at approximately 0.6 µmol cm-2 above which no increase in grayscale intensity was observed (data not shown). Additional experiments loading naphthol AS-D acetate onto cellulose filter papers indicated that measured grayscale intensities increased up to 1.5 µmol cm-2 but the signal intensity was lower than that for the naphthol AS-MX acetate substrate at all loadings. This suggests that either less naphthol AS-D acetate was adsorbed onto the filter surface (with the excess simply leaching into solution) or that more of the AS-MX form of the substrate was available for enzymatic hydrolysis at any given loading. Further experiments loading these acetate substrates and naphthol AS-MX butyrate onto Supor-450 filter membranes indicated no increase in signal intensity at loadings higher than 0.1 µmol cm-2. Again, this was either due to saturation of the polysulfone membrane at lower loadings or possibly the polysulfone structure trapping the adsorbed enzyme substrate, rendering it unavailable for hydrolysis. Polysulfone is not susceptible to microbial degradation and represents a more stable material for longterm deployments in sediments, but the saturation of the filters at such low substrate loadings decreases the dynamic range of the technique to an extent where spatial differentiation of esterase activities is not readily feasible. These effects were not investigated further. Of the filters and substrates evaluated in this work, the most robust system for construction of the enzyme activity probes was a cotton cellulose filter support loaded with naphthol AS-MX acetate. This system produced evenly loaded filters with appropriate dynamic range to map esterase activity in sediments. Color Development. The optimal Fast Red TR concentration for the development of filters loaded with 0.6 µmol cm-2 naphthol AS-MX acetate was equivalent to 4.7 µmol cm-2 (10 mL, 0.6 mg mL-1 per 4.8 cm2 filter membrane). This represented a 7.5-fold excess over the substrate concentration on a strictly molar basis (rate of hydrolysis notwithstanding). This ratio was used for all subsequent experiments. Increasing Fast Red concentration above this value did not increase the final grayscale intensities, possibly as a result of self-coupling of the azo compound at higher concentrations (during the development stage, a change from colorless to dark orange was observed in solutions above 1.0 mg mL-1). A wide range of diazonium salts, with different coupling energies and stabilities (19) are commercially available and could potentially be used for the further improvement of the technique. Method Calibration. A standard curve of grayscale intensities was prepared for a series of naphthol AS-MX loadings on cotton cellulose membrane filters. The unsubstituted naphthol has a hydroxyl group at the β position, thus leaving the R position free for direct coupling with the diazonium salt. It was assumed that all of the naphthol ASMX loaded onto the filter membranes was available to couple with Fast Red TR. Naphthol AS-MX is extremely insoluble in aqueous solution and has a maximum solubility in acetone of approximately 10 mM. This allowed a maximum loading of 0.2 µmol cm-2 on the filter papers. Gray scale intensities were measured for a series of filters loaded with 0.001 to 0.2 µmol cm-2 naphthol AS-MX and directly coupled to the Fast
TABLE 1. Reproducibility of Grayscale Intensity over a 300 mm2 Areaa mean within- betweengrayscale sample sample n intensity % RSD % RSD
treatment
enzyme hydrolysis 6 (Naphthol AS-MX acetate, 0.6 µmol cm-2) Naphthol AS-MX 6 (0.07 µmol cm-2) commercial colored paper 6
140.4
3.4
1.2
109.5
2.8
1.1
93.2
3.0
1.1
a
Comparison of the enzyme hydrolysis method with direct coupling of naphthol AS-MX and commercial colored paper. Grayscale intensity values are given for illustrative purposes only and are not comparable across the three treatments.
TABLE 2. Spatial Variability: 1 mm2 Measurements Made over the Surface of Filters Exposed to Different Enzyme Activities
FIGURE 1. (a) Calibration curve for esterase activity on 4.8 cm2 naphthol AS-MX acetate-treated cellulose filters; enzyme substrate loading 0.6 µmol cm-2, 24 h reaction time, Fast Red TR development 23.3 µmol per filter. Calculated naphthol AS-MX hydrolysis from eq 1. (b) Digital image of filter papers exposed to enzyme solutions. Red. There was a systematic, nonlinear relationship between the naphthol AS-MX loadings and grayscale intensity. The data were well fitted by
y ) a ln(bx)
(1)
where a and b are fitting constants a ) 26.1 and b ) 1330.5 and R2 ) 0.98. This nonlinear response suggested either a tail-off in sensitivity as more naphthol AS-MX was loaded onto the filter papers, or, at higher loadings, proportionately less of the naphthol reacted with the Fast Red to form the azo dye. However, the pixel values generated by a CCD device such as a flat bed scanner (and the Scion Image software) are not a measure of absorbance and are not necessarily linear with respect to concentration (23). This was confirmed by a simple experiment. Absorbance readings (λ ) 535 nm) for a series of naphthol AS-MX/Fast Red TR solutions in DMF (N,Ndimethylformamide) were measured on a spectrophotometer and were linear over the entire concentration range (R2 ) 0.99), but the measured mean grayscale intensities for the digitized images of each concentration produced a logarithmic curve similar to that achieved using the filter paper method. Thus, the observed nonlinearity was a property of the optical detection system in the flatbed scanner and not the filter paper methodology. Eq 1 was therefore used to transform measured grayscale intensities to concentration in all subsequent analyses. Response in Esterase Solutions. Membrane filters loaded with the enzyme substrate naphthol AS-MX acetate (0.6 µmol cm-2) were exposed to a series of enzyme solutions for 24 h, rinsed well, and developed with Fast Red TR. It was assumed that all the naphthol AS-MX acetate applied to the filters was available for enzymatic hydrolysis and that which was hydrolyzed would couple to the diazonium salt during the development stage. Figure 1 shows the relationship between the rate of product (naphthol AS-MX) formation per cm2, calculated from measured grayscale intensities using eq 1, and enzyme activity. A linear relationship (R2 ) 0.98) was observed for enzyme activities up to 1.0 unit of esterase mL-1 over the 24-h reaction period. These data suggest that the analytical range of the technique is in the region 0.1 to
relative enzyme activity (units mL-1) 0.5 0.2 0.1 0.05 0.02 0.01 0.005 0.002
n
nmol naphthol AS-MX cm-2 h-1 (mean)
betweensample %RSD
9 9 9 9 9 9 9 9
6.3 2.2 1.0 0.7 0.6 0.3 0.3 0.2
5.3 7.7 20.0 11.8 21.4 12.5 6.7 7.5
4.2 nmol naphthol AS-MX formed cm-2 h-1. This provides clear evidence that the technique is capable of providing a measurement of relative enzyme activity under controlled laboratory conditions. Reproducibility. The measurement of relative enzyme activity at precise spatial locations within sediments required that the application of the enzyme substrate was sufficiently uniform across the surface of the filter membrane. Table 1 shows the variability of results produced from enzymatic hydrolysis of the substrate and a post-coupling reaction, compared to the variability associated with direct coupling using the naphthol AS-MX parent compound and to that associated with a high-quality commercially dyed paper. Measurements were made over an area approximately 300 mm2 for six identical filters. The observed within-sample relative standard deviation was similar for the filters colored with naphthol AS-MX compounds (3-4% RSD) to that observed for commercial paper (3% RSD). Excellent reproducibly was observed between filters using this technique (Table 1). To determine more precisely the spatial resolution allowed by this method, 1 mm2 measurements were randomly taken over a set of filters (nine measurements per filter) which had been exposed to a series of increasing enzyme concentrations prior to development. These data are shown in Table 2 expressed as nmol of product formed cm-2 h-1, and indicate that the within-filter precision achieved in the technique is between 5 and 20% relative standard deviation. This was sufficient to allow acceptable differentiation of areas of relatively high and low enzyme activity at the mm scale. However, future improvements to the substrate application process may improve the within-probe spatial reproducibility. Laboratory Sediment Models. The spatial resolution of the relative enzyme activity probes was further tested by placing small amounts of homogenized sediment onto discrete areas of the filter surface. After 24-h reaction time, excellent definition of the enzyme activity in these areas of sediment was observed (Figure 2a). The principle aim of the VOL. 38, NO. 19, 2004 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
5137
FIGURE 2. Spatial resolution of sediment esterase activity: (a) sediment applied to discrete areas on the face of the probe; (b) digital image of the lateral structure obtained from in situ deployment in a laboratory model, 24 h contact time; and (c) origin of sediment enzyme activities (i) sediment, (ii) autoclaved sediment, (iii) porewater, (iv) autoclaved porewater, (v) sediment grains with porewater, and (vi) autoclaved sediment grains with porewater. method is to measure the spatial heterogeneity of sediment enzyme activity in situ and it is very difficult to assess the resolution of the technique for field deployments. A probe cannot be deployed exactly in the same place twice, and there may, in any case, be changes in enzyme activity in any given area of sediment with time. To try to overcome this difficulty, “model” sediments with areas of known high and low enzyme activity were constructed in microcosms in the laboratory. Alternate lateral bands of homogenized sediment (relatively high enzyme activity) and autoclaved sand (much lower enzyme activity) a few centimeters wide were placed in a glass beaker and autoclaved river water was carefully poured over the top. A thin strip of sediment was allowed to collect across the surface of the sand. Figure 2b shows the digital image produced from a probe which had been deployed for 24 h in a lateral sediment model. The differing enzyme activity associated with the model features can be distinguished clearly, as can the sediment water interface and the relatively low enzyme activity associated with the overlying water. A product formation rate of 0.3-0.4 nmol naphthol AS-MX cm-2 h-1 was observed on areas of the probe exposed to the sediment, but a rate of only 0.1 nmol naphthol AS-MX cm-2 h-1 (equivalent to the background value achieved in deionized water) was obtained for the autoclaved sand and water. To verify that the observed activity in the sediment was a result of enzymatic processes, homogenized sediment overlaid with river water was autoclaved at 121 °C for 20 min to remove biological activity. No substrate hydrolysis above the background value was observed on probes deployed for 24 h in these sterilized sediments, confirming that the product formation associated with the natural sediment was indeed biological and was not a result of abiotic hydrolysis reactions. Origin of Enzyme Activity. To differentiate between pore water and sediment-bound enzyme activities, pore waters were extracted by centrifuging and filtering a sample of sediment. The relative enzyme activities associated with 5138
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 38, NO. 19, 2004
FIGURE 3. Sediment esterase activity (Hacking River NSW, autumn 2003), measured as relative substrate hydrolysis (nmol cm-2 h-1) on the enzyme probe assembly as a function of sediment depth, 24 h deployment: ([) mm scale analysis, the vertical line on the digital image indicates the measured profile; (9) average values over the entire width of the probe for discrete 5-mm vertical sections. porewater, autoclaved porewater, individual sediment grains, and autoclaved individual sediment grains were determined separately. Despite the significant disturbance to both sediment chemistry and enzyme distribution caused by this experiment, higher activity was clearly associated with sediment grains (but not autoclaved sediment grains) than with porewaters (Figure 2c). No additional enzyme activity was observed when sediment grains were placed on a dry filter demonstrating that water flow or diffusion is required for sufficient contact between sediment-bound enzymes and the surface of the filter for hydrolysis of the substrate to occur. Mesocosm Deployments: Mapping Sediment Enzyme Activity in Situ. Examples of a vertical profile (Figure 3) and two-dimensional measurements (Figure 4) of relative sediment enzyme activity from probes deployed in laboratory mesocosms revealed considerable spatial heterogeneity in some aquatic sediments. A probe deployed in an intact freshwater sediment mesocosm from the Hacking River in NSW (Autumn 2003; Figure 3) showed an unusually large “hotspot” of enzyme activity approximately 5-10 mm wide and about 10 mm below the surface of the sediment. An area of enzyme activity this large is atypical in the sediment probes we have so far deployed in freshwater systems, but nevertheless this serves to illustrate the occurrence of lateral structure in microbial activity. A 1-mm wide vertical profile was measured through the area of high enzyme activity and the resulting product-concentration depth profile was plotted at mm intervals. This clearly shows a spike of increased product formation; rising to about 2.5 nmol naphthol AS-MX cm-2 h-1, formed about 10 mm below the sediment surface. To generate data on the same spatial scale as a traditional slicing method average intensity values were measured over the entire width of the probe at 0.5-cm vertical intervals; this gave a maximum of only 0.5 nmol product cm-2 h-1 in approximately the same position as the 1-mm wide profile. Thus, the activity in the large “hotspot” of enzyme activity would not be identified to the same degree by a traditional core-slicing method. Further evidence for small-scale lateral heterogeneity in sediment esterase activity is provided in Figure 4. In this example 4-mm2 measurements of intensity values were generated over the entire area of a
FIGURE 4. Two-dimensional map of esterase activity in a sediment mesocosm from Five Dock Bay, NSW, Autumn 2003. The sedimentwater interface (SWI) is represented by 0 on the depth profile; positive depths indicate positions above the SWI. probe deployed in a mesocosm sample from the Port Jackson Estuary NSW (Autumn 2003). Several hotspots of product formation were identified on the exposed probe (e.g., points a and b in Figure 4), and areas of lower enzyme activity associated with bioturbation at the sediment surface (e.g., point c) are also visible. The current resolution of this technique is at the millimeter scale. At this scale, enzyme distribution is not measured at the single cell or colony level. The higher activity “hotspots” are associated with solid sediment structures however, which reflect microbial distribution if most cells are attached to sediment grains (15). This is also demonstrated by consideration of the relatively low enzyme activities associated with sediment pores, e.g., areas of visible bioturbation within the sediment. The data shown in Figure 4 suggest that, in this sediment, hotspots of activity are not caused by pores in the sediment matrix allowing higher rates of water flow and extracellular enzyme diffusion to the probe surface. This is the first reported method for measuring the 2Dspatial distribution of microbial activity in situ in aquatic sediments. The methodology is very simple and the probes may be rapidly and inexpensively constructed in the laboratory. Deployment of the sediment probes in situ in sediments over time periods of up to 24 h allows for an integrated profile of enzyme activity at spatially distinct locations over time. The probes have been designed to cause minimal disturbance to the sediment and are
