Phosphatase activity in arctic tundra soils disturbed by vehicles

Soil Biol. Biochem. Vol. 22. No. 6, pp. 853-858, Printed in Great Britain. All rights reserved

PHOSPHATASE

0038-0717/W $3.00 + 0.00

1990

Copyright(Q 1990 Pergamon Press plc

ACTIVITY DISTURBED SUE A.

IN ARCTIC TUNDRA BY VEHICLES

SOILS

and JOHN L. NWL*

HERBEIN

Microbiology and Immunology Section, Department of Biology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, U.S.A.

Sammary-Increased productivity and improved nutrient status of plants in arctic tundra soils disturbed by vehicles has been attributed to increased availability of P that may be related to mineralization of organic P by soil phosphatase enxymes. Phosphomonoesterase and phosphodiesterase activities were examined in disturbed and undisturbed organic arctic soils collected from two sites: a wet site approaching water saturation and a drier, well drained site. Only acid phosphomoesterase activity was detected, while an acid to neutral phosphodiesterase was found. Phosphomonoesterase activity was 20- to 30-fold greater in wet tundra soil than in corresponding drier soil and phosphodieaterase activity was 25- and 35-times greater in soils from the wet site than in soils from the dry site. Vehicle disturbance of the wet tundra soil ecosystem caused a significant decrease in phosphomonoesterase and phosphodiesterase activities. In contrast, enxyme activities were not significantly affected by vehicle disturbance in soils from the drier site. The results show that maximal enxymc activity occurs at the pH of the natural ecosystem. Based upon the amount of enzyme activity, our results support the conclusion than phosphatases may play a significant role in the nutrient cycling process of P in arctic tundra soils. However, the potential for increased P availability in soils disturbed by vehicles could not be attributed to increased soil phosphatase activity.

INTRODUCTION Tracked vehicle passage over arctic tundra causes thinning of the vegetation and litter allowing for increased thermal conductivity of the organic horizons resulting in a warmer soil, deeper thaw and soil subsidence (Bliss and Wein, 1972; Babb and Bliss, 1974; Haag and Bliss, 1974; Gersper and Challinor, 1975; Challinor and Gersper, 1975). Additional heat from the influx of ground water increases depth of thaw and plant nutrient concentrations are affected by the altered soil environment (Wein and Bliss, 1974). Higher concentrations of potentially-available nutrients, particularly P, are found in soil beneath vehicle tracks compared with soil in the adjacent undisturbed tundra (Chahinor and Gersper, 1975; Chapin and Shaver, 1981). The content of N and P in the above-ground standing crop also changes (Chapin and van Cleve, 1978; Chapin et al., 1978, 1979). A feature of the total P budget in tundra soils is the large size of the organic P pool as compared to the mineral P of which only cu 12% is exchangeable (Chapin et al., 1978). The atmospheric input of nutrients is quite low and the cold temperature, presence of permafrost that impedes drainage of soils, and the dominance of anaerobic conditions that produce an acid environment, slows the recycling of organically-bound nutrients. However, microbial decomposition of organic matter may be the major route by which soil P is recycled in some arctic soils (Hill and Tedrow, 1961). Although soil enzyme activity in soils has received considerable attention (Bums, 1978), limited information exists about the activity of enzymes in arctic *To whom all correspondance should be addressed.

soils. Linkins and Neal (1982) investigated soil cellulase, chitinase and protease activity in a microsuccession of plants (Fletcher and Shaver, 1982) associated with tussock tundra formed by Eriophorum oaginaturn. Those soils associated with tussocks with the greatest plant diversity exhibited the greatest total enzyme activity; those with fewer plants had a lower rate of enzyme activity. They concluded that not only the dominant plant species, but also the nature of plant litter and associated soil environmental conditions, were differentially affecting cellulase, protease and to lesser extent, chitinase activity. Neal (1982) studied phosphomonoesterase and sulphatase enzyme activity in arctic soils subjected to an environmental gradient established by snow patch melt and found that rate of enzyme activity could be correlated with the distinct zones of vegetation that occurred within the gradient. Although soil moisture and water movement could be implicated in altering the amount of enzyme activity, their exact role was not delineated. Sulphatase activity in arctic soils (Neal and Herbein, 1983) was found to be significantly less in soils perturbed by tracked vehicles than that in adjacent undisturbed soils. The mineralization of organic S in disturbed tundra soils was hypothesized as being controlled by nutrient influx associated with water movement, altering sulphatase activity to a rate consistent with the supply of the mineralized S. Chapin and Shaver (1981) postulated that increased productivity of disturbed arctic soils may be related to altered rates of nutrient cycling and nutrient availability in the soils. They concluded that increased temperature and depth of thaw in the vehicle-tracked soils was not sufficient to explain the improved nutrient status and growth of tundra plants in the disturbed soils. Based upon their study, we

853

SUEA. HERBEINand JOHN L. NML

854

hypothesized that increased P availability in the vehicle-tracked soils could be attributed, in part, to increased mineralization of organic P by soil phosphatases. Our purpose was to determine phosphomonoesterase and phosphodiesterase activity in arctic tundra soils collected from under tussocks in vehicletracked and adjacent undisturbed sites. The amount of enzymatic activity was determined over a broad pH range to ascertain if maximum enzyme activity could be correlated with soil pH. MATERIALS AND METHODS

Site and soil description Arctic sampling sites were located in upland tussock tundra east of Slope Mountain (68”43’N, 149”OO’W, 655 m elevation) adjacent to the TransAlaskan Pipeline Haulroad, 175 km south of Prudoe Bay, Alaska. Approximately 9 yr before sampling, tracked vehicles had partially removed the vegetative mat and compacted the tundra surface. The vehicle track extends up a 5” north facing slope from wet tundra dominated by Eriophorum angustiforium and Carex aquatilis, through less wet tundra dominated by E. vaginatum, to dry tussock tundra dominated by E. vaginatum and L.&m paiustra sp. decumbens. Water flow is channeled by the vehicle track. Two sites were chosen for study: (1) a wet vehicle track and undisturbed tundra adjacent to it which approached water saturation; and (2) a drier vehicle track in a well-drained area further up the slope and its adjacent undisturbed tundra. Soils from Site 1 were tentatively classed as Histic, Pergelic Cryaquepts and soils from Site 2 were classed as Pergelic Cryaquepts (K. R. Everets, personal communication). Some general characteristics of the vehicle-tracked and uncompacted soils are shown in Table 1. A detailed description of the characteristics of the soil beneath the tussocks is reported by Chapin and Shaver (1981). Analytical methodr Random samples of arctic soil, following the procedures described by Petersen and Calvin (1965), were collected in triplicate from disturbed and undisturbed areas at each site. Because soil horizons were difficult to accurately determine, samples were Table I. Selected properties of vehiik-track

UH’

Wer sire Vehick-track Undisturbed Dry sire Vehicle-track Undisturbed

4.7 4.7

disturbed and undisturbed arctic tundra soils

(mscm-‘1

Bulk dcnsityc Imacm-‘)

Organic matte* (% dry wt)

Available phosphor& (mM)

574 401

87.2 124.7

83.1 80.0

0.17 0.10

42 35

105.8 87.5

82.2 79.8

1.53 1.50

Moist& Soil

collected to a depth of 10 cm from under tussocks in the vehicle tracks and adjacent uncompacted tundra. For the purpose of our report, soil is defined as the organic matrix beneath the tussocks. Soil samples were collected during the second week of August, sealed in plastic bags, frozen and maintained at -25°C until processing. All white- or yellow-white live roots were carefully removed from the samples before storage. Each soil sample was divided into three subsamples of approximate equal weight (Petersen and Calvin, 1965), placed in liquid N, and ground while frozen in a steel friction grinder to < 2 mm, thoroughly mixed, and stored at -25°C in sealed plastic bags. Tests with other arctic soils indicated that storage at -25°C was sufficient to severely limit microbial respiration and loss of phosphatase activity (Neal, 1982). Phosphomonoesterase activity was measured as described by Tabatabai and Bremner (1969) and phosphodiesterase activity assayed according to the procedure of Browman and Tabatabai (1978). The use of NaOH to extract p-nitrophenol from some organic soils has been reported to yield highly variable blanks (Sarathchandra and Perrott, 1981). We found that the recovery of known concentrations of p-nitrophenol from the soils used in this study was quantitative and the use of NaOH as an extractant did not interfere with the accurate calorimetric determination of p-nitrophenol. The influence of pH on the rate of phosphomonoesterase enzyme activity was determined by adding modified universal buffer (MUB) (Tabatabai and Bremner, 1969) adjusted to a specific pH. Before the addition of substrate, the soil buffer suspension was readjusted, if required, with either 0.1 N HCl or 0.5 N NaOH to the intial buffer pH. This ensured that all soils, regardless of their initial pH, were assayed under near identical conditions of soil buffer pH. The same procedure was followed for the measurement of phosphodiesteraseactivity, except 50 mM tris(hydroxymethyl)aminomethane (THAM) buffer was used instead of MUB. Phosphomonoesterase activities were measured at a pH of 1 through 12 and phosphodiesterase activities determined at a pH of 2 through 12. All enzyme activity measurements were done in triplicate as static 1 h exposures at 20°C. Selective monitoring of change in pH during the 1 h exposure showed a change of < 0.1 pH units. To ensure maximum colour development of p-nitrophenol, final filtrate pH was maintained at 8.5 or higher. Filtrate dilution, when required, was made with pH 8.5 MUB buffer for phosphomonoesterase assays or pH 10.0

‘Determined on a 1:I soil:water paste (McL.ean. 1982). bGravimetric determination (Gardner. l%S). ‘Courtesy of F. S. Chapin 111 (pcraonal communication). “Dichromate method (Alliin, 1965). ‘Acid-extractable. plant-rvailabk phosphorus (Donahue and Gettier. 1980).

Phosphatase activity in arctic soils

2

4

6

6

IO

855

I2

SOIL BUFFER SUSPENSION pH

SOIL BUFFER SUSPENSION pH

Fig. I. Effect of pH of soil buffer suspension upon phosphomonoesterase activity in (A) wet vehicle tracked disturbed (A) and undisturbed (0) and (B) drier, drained vehicle tracked disturbed (A) and undisturbed (0) arctic tundra soils. SD of each mean is represented as a vertical bar.

0.1 M THAM buffer for phosphodiesterase assays. The SD of the sample means was calculated according to accepted methods (Zar, 1974). RESULTS

Phosphomonoesterases (phosphoric monoester hydrolases) in soil are believed to catalyse the hydrolytic removal of phosphate groups from inositol phosphates, nucleotides, sugar phosphates and glycerophosphates (Speir and Ross, 1978). Phosphomonoesterase activity in the organic soils examined from both the wet site and dry site, disturbed and undisturbed, was active over a pH range of 2-12 (Fig. 1). Enzyme activity was found to be maximal within an acid pH range of 4-6, with apparent optimal activity at pH 5 in soils sampled from Site 1 and Site 2 (Fig. lA, B). Alkaline phosphomonoesterase activity was not evident. At soil buffer pH where phosphomonoesterase activity was maximal, phosphomonoesterase activity in wet tundra soil, Site 1, was 20- to 25-fold greater than that in the corresponding drier tundra soil, Site 2 (Table 2). Enzyme activity in the wet undisturbed arctic soil was found to be twice that in the wet vehicle-track disturbed soil (Fig. lA, Table 2). However, the amount of phosphomonoesterase activity was not significantly different between the drier vehicle-track disturbed soil and adjacent undisturbed soil (Fig. lB, Table 2). The pH optima for maximum enzymatic hydrolysis were found to be closely related to measured pH of the soils (Table 2). Phosphodiesterase (phosphoric diester hydrolases) catalyses the hydrolysis of ribo- and deoxyribonucleic acids and phospholipids in soil (Speir and Ross, 1978). Although the enzyme was found to be active from pH 2 to 12 (Fig. 2), maximum activity was found to be optimal between pH 4 and 7, indicating the presence of acid or neutral phosphodiesterases. In the soils sampled from the wet site (Fig. 2A), apparent enzyme activity was maximal at pH 7, while apparent activity in the dry site soils was maximal at

a pH of 5-6 (Fig. 2B). At the soil buffer pH where phosphodiesterase activity was found maximal, phosphodiesterase activity was 2% to 3%times greater in the wet tundra soils than in the corresponding drier tundra soils (Table 2). Vehicle disturbance had little effect upon the amount of enzyme activity in soil samples from the drier vehicle track and adjacent undisturbed soil and essentially no difference was found in the amount of activity over the range of soil buffer pH employed. In contrast, phosphodiesterase enzyme activity was 1.7~times greater in undisturbed soil compared to vehicle-disturbed soil sampled from the wet site. The pH of the tundra soils, whether disturbed or undisturbed, corresponded to a soil buffer pH at which phosphodiesterase activity was maximum or near maximum (Table 2). Compared to phosphomonoesterase activity, the amount of phosphodiesterase activity was 10 times less in the wet disturbed and undisturbed soils, as well as in the drier disturbed and undisturbed soils, suggesting that phosphomonoesterase is the dominant enzyme involved in the mineralization of organic P in these tundra soils. DISCUSSION

Soil pH is one of the most indicative measurements of a soil. Whether the soil is acidic, neutral or basic Table 2. Phosphomoncstcrasc and phosphodicsterasc activities @g of p-nitrophcnol g-’ soil.h-’ f SD) at optimal pH and soil pH in whick-track disturbed and undisturbed arctic tundra soils Phosphomoncstenw activity Soil

?Hmal

Wer sire Vehicle-track Undisturbed

2592 f M8 4240*173

Dry sire Vehicle-track Undisturbed

128 f 56 172kSO

Pbosphodicstcrasc activity

Soil PH

Optimal PH

Soil

2570 f 236 408Okl64

28s f 34 444i84

263 f 31 416*67

IIS* 164*38

13f 12*3

I

PH

II f I II *2

SUE A. HERESINand JOHNL. NEAL

856

A

600

i c i= 24

s P

E * P

‘6

B

F

Inw 9

iid d

6

5 E

2

4

6

SOIL BUFFER

6

IO

SUSPENSION

I2 pH

i z h

2

4

6

SOIL BUFFER

6

IO

SUSPENSION

12 pH

Fig. 2. Effect of pH of soil buffer suspension upon pbosphodiesterase activity in (A) wet vehicle tracked disturbed (A) and undisturbed (0) and (B) drier, drained vehicle tracked disturbed (A) and undisturbed (0) arctic tundra soils. SD of each mean is represented as a vertical bar.

has much to do with solubility of various compounds, the relative bonding of ions to exchange sites, the activity of various microorganisms, and the enzymes found in the soil solution or complexed with the organic and clay constituents of soil (McLean, 1982). Changes in the H+ concentration influences the enzymes, substrates and enzyme cofactors by altering their ionization and solubility. The rates of enzymatic activity may exhibit marked changes resulting from fluctuations in soil pH (Frankenberger and Johanson, 1982; Tabatabai, 1982). In our study, distribution of phosphomonoesterase activity in soil was pH related (Fig. 1). Eivazi and Tabatabai (1977) and Juma and Tabatabai (1978) measured the response of phosphomonoesterases to pH in Iowa mineral soils utilizing the same Modified Universal Buffer system used in our study and found a distribution of both acid and alkaline phosphomonoesterases. Using a different buffer system, Halstead (1964) found two distinct activity peaks in an organic soil buffered at pH 5.0 and 9.5. In our study, only acid phosphomonoesterase activity was found. Similar results for organic soils of Galicia (NW Spain) were reported by Trasar and Gil-So&s (1987, 1988). They suggest the phosphomonoesterase enzymes have the capacity in the soil medium to adapt to the condition of the soils and perhaps the enzymatic reaction is catalysed by more than one enzyme of multiple forms of the same enzyme (Nannipieri et al., 1982). Soil pH also had an effect upon the distribution of phosphodiesterase enzyme activity. The acidic arctic soils have a phosphodiesterase activity pH optimum near 6, which was the optimum reported by Hayano (1977) for phosphodiesterase extracted from a forest soil and measured in TRIS-Maleate buffer. In comparison, the optimum pH for phosphodiesterase activity in four agriculture soils was reported to be near 8 (Browman and Tabatabai, 1978). Phosphomonoesterase catalysed organic P release to the inorganic form, as measured by the hydrolysis of p-nitrophenol phosphate to p-nitrophenol and

inorganic ._ . P, appeared to be potentially the major contnbutmg enzyme system carrying out the hydrolysis of organic P in the arctic soils. Phosphomonoesterase activity was lo-times greater than phosphodiesterase activity in the same soil. This may reflect the type and quantity of organic substrate available for mineralization. The results indicate that in these arctic soils, hydrolysis of organic P by phosphomonoesterase and phosphodiesterase ap proaches the maximum rate of hydrolysis at the pH of the soil. Phosphomonoesterase and phosphodiesterase activities were both found to be greater in arctic soils collected from the wet site than from the drier, more well-drained sites. The major ecological difference between the two sites was soil moisture and water movement. Experiments with enzymes bound to an artificial matrix (Engasser and Horvath, 1974) showed that when diffusion of substrate to bound enzymes was slow, the substrate. concentration in the microenvironment was lower than that in the macroenvironment. Tabatabai and Bremner (1971), Brams and McL,aren (1974) and Irving and Cosgrove (1976) have reported data that suggest reactions catalysed by soil enzymes may be diffusion limited. Increased soil moisture and water movement could facilitate substrate diffusion to and product diffusion away from the immobilized enzymes. Therefore, a moisture related change in the pattern of P cycling may partially explain the greater amount of phosphatase activity in the wet site soils. Phosphomonoesterase and phosphodiesterase activities in the arctic soils used in our study were found to be lower in wet, disturbed soils than in adjacent undisturbed tundra. Based upon our results, increased P availability in the vehicle-disturbed soils would not be directly related to increased phosphatase activity. The rate of organic P catalysis by phosphatase enzymes depends upon a number of interrelated soil properties, as well as the properties of the enzymes. Increased moisture flow into the vehicle track causes that soil to be more reduced

Phosphatase activity in arctic soils (lower redox potential) than the adjacent undisturbed tundra. Arctic soils apparently have a high iron content (Bunnell et al., 1975; Bare.1 and Barsdate, 1978) and ferrous iron was present in vehicle track soils at Slope Mountain, but absent from adjacent undisturbed soils (Chapin and Shaver, 1981). Pulford and Tabatabai (1988) found that waterlogging Iowa soils caused a general, but significant decrease in phosphatase activities. They suggest the reduced activities were due to inhibition by the reduced metal ions produced by waterlogging the soils. Reduced metal ions have been shown to be stong inhibitors of phosphatases in soils (Juma and Tabatabai, 1977; Stott et al., 1985). It is well known that while phosphate is absorbed strongly under conditions of high redox potential, a portion is desorbed under conditions of low redox potential. Desorption of absorbed inorganic P increases in response to increased soil temperature (Mack and Barber, 1960; Singh and Jones, 1977), a characteristic of vehicledisturbed soils attributed to increased water fiow into the track (Gersper and Challinor, 1975; Chapin and van Cleve, 1978; Chapin and Shaver, 1981). Orthophosphate acts as an inhibitor of soil phosphomonoesterase in mineral temperate soils (Juma and Tabatabai, 1978) and forest soils (Pang and Kolenko, 1986). The potential exists for increased concentrations of inorganic phosphate in wet vehicle track soils as result of continuous movement of water into the vehicle track by desorption of the inorganic P, and release of P into the soil solution when iron is reduced from ferric to the ferrous state. Our study suggests that, in arctic tundra soils, the amount of soil moisture may be a major factor in regulation of phosphatase catalysed mineralization of organic P and in the availability of mineralized inorganic phosphate. Possible constraints placed on phosphorus cycling in the organic tundra soils may be.: (1) the rate of diffusion of organic substrate to bound phosphatases; (2) the rate of diffusion of end product inhibitor away from the bound enzyme; and (3) the ratio of ferric to ferrous iron based upon the moisture dependent interaction of soil pH and redox potential. The more reduced vehicle-track soils could maintain a greater potential for release of insoluble inorganic phosphorus to soluble forms, thus meeting plant and microbial requirements at a lower phosphatase activity and reaction velocity. In turn, the availability and cycling of inorganic nutrients as mediated by phosphatase enzymes probably exert a measure of control on microbial activity, plant productivity and composition, in disturbed as well as undisturbed arctic soils. Acknowledgemenrs-We thank A. Bymes for technical assistance. This research was supported in part by a grant from the Virginia Polytechnic Institute and State University Agriculture and Forestry Core Research Program. We thank the Institute of Arctic Biology, University of Alaska, Fairbanks, Alaska, for logistical and field support by means of a U.S. Army Research Office Grant.

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