Microbial enzyme activities related to litter decomposition near a highway in a sub-tropical forest of North East India

Soil Bid. Biochem. Vol. 25, No. 12, pp. 1763-1770, Printedin Great Britain. All rightsre~rved

1993

Copyright 0

0038-07 I7/93 $6.00 + 0.00 1993 Pergamon Press Ltd

MICROBIAL ENZYME ACTIVITIES RELATED TO LITTER DECOMPOSITION NEAR A HIGHWAY IN A SUB-TROPICAL FOREST OF NORTH EAST INDIA S. R. Jos~r,’ G. D. WARMA** and R. R. MI~HRA* ‘St Anthony’s College, Shillong-793 001, Meghalaya, and *Department of Botany, North-Eastern Hill University, Shillong-793 014, Meghalaya, India (Accepted II June 1993)

Summary-Cellulose, amylase. and invertase activities were studied in extracts of leaf litters of Alms nepulensh and Pinus kesiyu during litter decomposition at a roadside (more polluted) and a non-roadside (less polluted) forest stand. Enzyme activities were considerably higher in litter at the less polluted than at the more polluted site. Cellulase and amylase activities showed a marked seasonal variation at both sites. Cellulase and amylase activities increased during litter decomposition, whereas invertase activity was higher at the begining of litter decomposition. Invertase activity correlated positively with litter soluble sugars. Cellulase and amylase activities, but not invertase activity, were correlated significantly with numbers of fungi and bacteria.

INTRODUCTION The North-Eastern region of India is undergoing industrial development at an increasingly rapid rate. Roads form the main system of communication owing to the hilly topography of the region. Automobiles discharge a number of gaseous and metal pollutants during the combustion of petroleum fuels. Human activities such as stone grinding, road construction and sand milling also increase the atmospheric dust and heavy-metal contaminant level (Smith, 1976; Tyler, 1984; Alloway, 1990). These activities may affect the composition and activity of microorganisms in litter and change the rates of mineralization and immobilization near highways. Cellulose, hemicellulose and lignin are the major components of forest litter, comprising 50-80% of the dry mass (Swift et al., 1979). Cellulose and hemicellulose are recalcitrant products added to soil through plant remains and must be transformed into soluble substances prior to microbial assimilation through extracellular enzymes (Bums, 1978; Ross and Speir, 1979). Cellulose is a major structural component of litter and therefore is a vital energy source for the microbes associated with litter degradation (Sinsabaugh et al., 1981). Its hydrolysis into glucose is achieved by cellulase-enzyme complexes produced by fungi (Miele and Linkins, 1978). Sucrose is a major soluble carbohydrate in plant tissues. The mechanism of sucrose breakdown is an important process as it forms the major source for carbon allocation. Sucrose is solubilized by invertase, which can be either acidic or alkaline in nature (Spalding, 1980). *Author for correspondence.

Starch is another common compound within most plant tissues which increases during active photosynthesis and decreases as it is enzymatically converted into sugars. Amylases hydrolyse starch and make its carbon available for translocation. Cellulases are the most extensively studied enzyme system in plant litter due to their ubiquity (Eriksson and Wood, 1985; Sinsabaugh and Linkins, 1989; Linkins et al., 1990a, b). Very little information is available on amylase and invertase, in spite of their role in the hydrolysis of sugars and starch, respectively (Ali and Kalyansundarum, 1991; Geissman er al., 1991; Vainstein and Peberdy, 1991). Production of extracellular enzymes (cellulase, amylase and invertase) by microbes during litter degradation may be influenced by temperature, moisture, pH and the substrate involved (Linkins et al., 1984; Hayano, 1986; Sinsabaugh and Linkins, 1987), but the effect of environmental pollution on litter microbial enzymes has not been studied. Therefore, the aim of our study was to determine the activities of the microbial enzymes during tree-litter decomposition in polluted and unpolluted forest stands of North-East India. MATERIALS AND METHODS

Study site

The study was carried out at the Shillong-Jowai highway (altitude 1700-1730 m M.S.L.), located between latitude 25”34’ N and longitude 91”46’ E in the East Khasi Hills district of Meghalaya. Two study areas were selected, one a polluted site, at National Highway No. 44 with a high traffic density (8000-9000 vehicles/day), and the other a relatively unpolluted site about 500 m away from the roadside. 1763

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S. R. JOSHIet

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Litter &composition and microbial populations

While selecting the sites, care was taken to ensure similarity in topography and both the surfaces were flat. The polluted site was 15 m away from the road. The forest was similar at both the sites with regard to vegatational composition, tree height, extent of canopy closure, depth of forest floor and the microenvironmental conditions. The soil of the sites was a sandy loam of laterite (oxisols) and acidic in nature. The climate is typically subtropical monsoonal, with the south-west monsoon and north-east winter winds influencing the weather conditions of the area. The climate of the study area remains warm and moist from May to September due to the south-west monsoon and cool and dry from December to February. The monthly rainfall ranged from 2.1 to 421.2mm during 1990 and 0.1 to 574mm in 1991. The average minimum and maximum temperature varied from 7.1 to 18.o”C and 15.6 to 24.3”C, respectively, in 1990 and 4.8 to 18.4”C and 14.0 to 24.6”C, respectively, during 1991. The relative humidity varied from 59 to 92% in 1990, and 56 and 93% during 1991 (Fig. 1). Pinus kesiya and Alnus nepalensis were dominant and codominant species selected for the present investigation.

The litter comprised only of recently fallen leaves and a common pool of A. nepalensis and P. kesiya litter was used for both sites. Air-dried intact litter (10 g) of each plant species was kept in nylon bags (size 20 x 20 cm, mesh size 1 mm) and spread randomly on the forest floor for decomposition (Bocock et al., 1960). The approximate total area of the forest floor used for spreading out the litter bags at each site extended to 2 ha. Five litter bags were collected every month and brought to the laboratory to assess the rates of decomposition, microbial populations and enzyme activities. Enumeration of litter fwgal and bacterial populations The dilution plate technique (Waksman, 1922) was used to count the total number of fungi and bacteria in the decomposing leaf litters. Determination of mohture content and pH

The moisture content of the decomposing litter was determined by drying 1 g of litter to constant weight at 80°C and calculated on a moist weight basis.

q Rainfall Min. temp. Max. temp. Relative humidity

M w u

600

/

h

NJMM 1989

L i N

1990 SAMPLING

J

PERIOD

M

I

J

1991 ( MONTHS)

Fig. 1. Monthly variation in rainfall, ambient temperature (maximum and minimum) and relative humidity of the study site.

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Litter microbial activities near a highway

160 -

al

30 0

J

M

M,g;o

S

N J

M,gMg, J

S

SAMPLING

D

J

M M,gJ90 S

N J

M,gy,

J

S

D

PERIOD (MONTHS)

Fig. 2. Monthly variation in the number of viable fungal and bacterial propagules of different leaf litters at the polluted (PS) and unpolluted (UPS) sites. AN = A. neppalensisand PK = P. kesiyn.

The pH was determined by grinding a suspension of litter in double distilled water (1: 5 w/v) and reading after 1 h with an electronic pH meter (Systronics, India). Determination nitrogen

of cellulose,

total

sugars

and total

seived ( < 0.2 mm) litter samples were used to determine cellulose, total sugars and total N. Cellulose and lignin were estimated by Jermyn’s (1955) method, and total sugars were determined by the method of Mahadevan and Sridhar (1982). Total nitrogen was estimated by the microKjeldahl method (Allen, 1974). Three replicates were analysed for each sample. Powdered

and

up to 50 ml with double distilled water. The extent of metal binding by filters was checked using metals stock solutions and was negligible (Proctor et al., 1980). Samples were analysed on a Perkin-Elmer 2380 Atomic Absorption Spectrophotometer after appropriate dilutions. Extraction

of enzymes

Cellulase, amylase and invertase activities were estimated by the technique of Spalding (1977). The litter was cleaned to remove adhering soil particles, and 5 g were transferred into a Waring blender and ground with 100 ml of chilled acetate buffer (pH 5.5) for 1 min. The homogenate was centrifuged at 9400g at 2°C for 20 min, and the supernatant filtered through a Whatman No. 1 filter paper.

Analysis of lead, zinc, copper and cadmium Three replicates, each of about 2g, oven dried litters were milled and weighed separately at each sampling. 0.2 g of the sample was digested in 20 ml

concentrated

HNO,. Extracts were filtered and made

Enzyme assays

For the enzyme assays, 1 ml of substrate solution and 2 ml of enzyme extract were reacted at 37 + 1°C for 2 h in a test tube. The substrates were 3%

1766

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R. JOSHI et al.

AN -ups WPS

o-013 P: 0.05

LSD:

LSD: P: 0.009 0. 05

P: 0.05

0,

* 1 I JMMJSNJMMJS 1990

I

I

*

I

I

*

I

1991 SAMPLING

-t D

o-

J

M19MgOJ

SN

-EUPS PS

J

MMJ 1991

S

PERIOD (MONTHS)

Fig. 3. Monthly variation in cellulase and amylase activity of different leaf litters at the polluted (PS) and unpolluted (UPS) sites. AN = A. nepalensis and PK = P. kesiya.

carboxymethylcellulose, Na-salt (Sigma) for cellulase, 6% soluble starch (Sigma) for amylase and 6% sucrose (Sigma) for invertase; they were dissolved in the same acetate buffer as that used for grinding the litter. The reducing sugars thus formed were determined by the dinitrosalicylic acid method (Miller, 1972), by measuring the absorbance at 575 nm (Hitachi-220). Enzyme activity was expressed as reducing sugars formed g- ’ litter h- I.

RESULTS

Microbial populations

The fungal and bacterial populations were at a minimum at the begining of leaf litter decomposition, but increased rapidly as the decomposition progressed, and then decreased towards the end of the process. Bacterial populations were higher in P. kesiya than in A. nepalensis litter, whereas fungal populations were higher in A. nepalensis litter. Micro-

Table 1. Correlations coefficients (r) of cellulase, amylase and invertase activities with the fungal population moisture content, pH, total nitrogen, weight loss and cellulose and total sugars contents of A. mpalemis litter Unpolluted site

Polluted site Sources of variation Fungal population Bacterial population Moisture content PH Total nitrogen Weight loss Cellulose Total sugars ‘P < 0.05; l*P < 0.01; **+p

DF 14 14 14 14 14 14 14 14 < 0.001.

Cellulase 0.850*** 0.592’ 0.752*** 0.397 -0.172 0.342 -0.326 -

Amylase 0.966*** 0.606 0.888*** 0.657** 0.126 0.060 -

Invertase

Cellulase

Amylase

Invertase

-0.021 -0.371 0.065 0.377 0.941*** -0.956*** 0.954***

0.640** 0.499’ 0.595’ 0.211 -0.413 0.548. -0.499’ -

o.ss1*** 0.73 I ** 0.835*** 0.521; -0.119 0.250 -

0.015 -0.153 -0.013 0.435 0.935*** -0.971*** 0.958***

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Litter microbial activities near a highway

Table 2. Correlations coefficients(r) of cellulasa, amylase and invertase activities with the fungal population, bacterial populations, moisture content, pH, total nitrogen, weight loss and cellulose and total sugars contents of P. kesiya litter Unpolluted site

Polluted site sources of variation

DF

Cellulase

Amylase

Invertase

Cellulase

Amylase

lnvertase

Fungal population Bacterial population Moisture content PH Total N Weight loss Cellulose Total sugars

22 22 22 22 22 22 22 22

0.651.‘. 0.693*** 0.713*** 0.169 -0.202 0.329 -0.423; -

0.528.. 0.530.1 0.614** 0.320 - 0.074 0.173 -

-0.047 -0.198 0.091 0.085 0.771*** -0.848***

0.297 0.262 0.344 0.037 0.024 0.046 -0.116 -

0.549** 0.383 0.583** 0.269 0.175 -0.004

-0.0825 -0.328 0.053 0.219 0.828*** -0.884*** 0.922***

O&7***

-

‘P < 0.05; **p < 0.01; l**p < 0.001.

bial populations were higher in these leaf litters at the unpolluted site than at the more polluted one (Fig. 2). Cellulase activity Extractable cellulase activity (Fig. 3). was higher in alder leaf litter than in the pine. In both kinds of litter, cellulase activity was lowest at the beginning, and increased as decomposition progressed. Seasonal variation in cellulase activity was observed, with a peak in May-June and minimum during winter. Cellulase activity was correlated positively with fungal and bacterial numbers and litter moisture content, and negatively with the cellulose content of the litters (Tables 1 and 2). Amylase activity Amylase activity was also generally higher in alder than in the pine litter. It increased during litter decomposition and showed a marked seasonal variation, with values generally highest in June and lowest during the winter months (Fig. 3). Amylase activity was correlated significantly with fungal and bacterial numbers and moisture contents and, in some samples, with the pH of the litters (Tables 1 and 2). Invertase activity In contrast to cellulase and amylase, invertase activity was high at the beginning of litter decomposition and, after a few weeks, decreased as decomposition progressed (Fig. 4). Activity was again higher in A. nepalensis than in P. kesiya litter. Invertase activity was significantly correlated with the total sugars and nitrogen contents of the litters (Tables 1 and 2). Generally, the activities of cellulase, amylase and invertase were higher in leaf litters at the unpolluted than at the polluted site. DISCUSSION The negative correlation between cellulase activity and the weight of cellulose remaining suggests that the amount of cellulose acts as a limiting factor for

the activity of cellulase. The high activity in A. nepalensis litter may be attributed to its high cellulose and nitrogen contents [Table 3(a)]. The increased cellulase activity in May-June was significantly correlated with fungal and bacterial numbers (Kshattriya et al., 1992). Cellulase activity was correlated positively, and generally significantly, with the moisture content of the litter, suggesting the favourable role of moisture in the synthesis of cellulase (Speir and Ross, 1981; Gressel et al., 1983). Ross (1981) has also observed lower invertase activity in litter organic horizons than in fresh leaves. The high invertase activity during the initial stages of decomposition was associated with high contents of soluble sugars in the litter. The low activity of invertase towards the end of decomposition may have been resulted from the exhaustion of specific substrates and lower numbers of microbes. The changes in amylase activity during litter decomposition are attributed to changes in the numbers of microorganisms, confirming the probable microbial origin of this enzyme (Ross and Roberts, 1973). The different amounts of cellulase, amylase and invertase extracted from the different leaf litters are attributed to differences in leaf chemistry (Spalding, 1980) and the nutritional requirements and preferences of the heterotrophs thriving on the litters (Sinsabaugh and Linkins, 1987). The low concentrations of these enzymes in P. kesiya litter may be attributed to high amounts of phenolic compounds, which may be generated by degradation of lignins and inhibit the production of microbial enzymes (Mandels and Reese, 1965; Benoit and Starkey, 1968). The lower production of cellulase, amylase and invertase in leaf litters at the polluted site than at the unpolluted site is attributed to the lower microbial populations at the former than at the latter site. The initial phase of decomposition is characterized by large amounts of soluble constituents of litter which are assimilable by the microbiota (Linkins et al., 199Oa). Different amounts of litter constituents remaining after different periods of decomposition at the two sites, as well as altered microbiota due to highway pollution, may have resulted in the differences

S. R. Josm et al.

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AN o--o UPS

LSD: 0.013

PK M

UPS

UPS

O-2 -

0

’ J

I M

I I M J 1990

I 1 # 1 MMJS 1991 SAMPLING PERIOD (MONTHS 1 1 S

I N

1 J

t II

Fig. 4. Monthly variation in invertase activity of different leaf litters at the polluted (PS) and unpolluted (UPS) sites. AN = A. nepulensis and PK = P. kesiyo.

in activity of these enzymes at the polluted and unpolluted sites. The results of our investigation suggest that microbial enzyme activities during litter decomposition are influenced to a great extent by changes in the micro-

bial populations caused by human activities and pollution near highways. This resulted in the slow rate of litter decomposition at the polluted site due to the higher amounts of soil pollutants near to the highway [Tables 3(a), (b)].

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Litter microbial activities near a highway

Table 3(a). Cellulose, lignin, total sugars, nitrogen and weight remaining of decomposing litters at two sites during initial and final stage of the study Litter components Leaf litters

Site

A. nepatensis

Polluted Unpolluted

P.

kesiya

Polluted Unpolluted

stage of study

Cellulose (%)

Initial Final Initial Final

43 14 43 10

Initial Final Initial Final

34 14 34 8

Lignin (%)

Total sugars tig IOOmg-‘)

Total N (%)

Weight remaining (%)

19 22 19 20

232 46 232 40

1.9 0.72 1.9 0.42

100 21.2 100 11.8

26 36 26 29

168 50 168 40

0.64 0.44 0.64 0.26

100 30 100 15.5

All data mean of three replicates. Table 3(b). Average concentration

of metals on decomposing litters at two sites

A. nepolensis M&dS

019 g-‘)

Polluted site

Lead Zinc Copper Cadmium

120 230 40 4

P.

Unpolluted site

Polluted site

20 SO 16 0.5

kesiya

Unpolluted site

190 260 36 4

30 70 12 0.6

All data mean of 16 and 24 sampling analysis of alder and pine litters respectively.

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