Microb Ecol (1993) 25:287-304

MICROBIAL ECOLOGYInc. © 1993Springer-Verlag New York

Decomposition of 14C-labeled Cellulose Substrates in Litter and Soil from a Beechwood on Limestone S. Scheu, 1 S. Wirth, 2 and U. Eberhardt 3 lII Zoologisches Institut, Abteilung Okologie, Berliner Strasse 28, W-3400 G6ttingen, F.R. Germany; 2Institut f/Jr Bodenforschung, ZALF Mtincheberg, Wilhelm-Pieck-Str. 72, O-1278 Mfincheberg, F.R. Germany; and 3Isotopenlaboratorium ffir biologische und medizinische Forschung, Burckhardtweg 2, W-3400 G6ttingen, F.R. Germany Received: September 22, 1992; Revised: January 19, 1993

Abstract. The decomposition of three different 14C-labeled cellulose substrates (plant holocellulose, plant cellulose prepared from 14C-labeled beech wood (Fagus sylvatica) and bacterial cellulose produced by Acetobacter xylinum) in samples from the litter and mineral soil layer of a beechwood on limestone was studied. In a long-term (154 day) experiment, mineralization of cellulose materials, production of 14C-labeled water-soluble compounds, and incorporation of 14C in microbial biomass was in the order Acetobacter cellulose > holocellulose > plant cellulose in both litter and soil. In general, mineralization of cellulose, production of ~4C-labeled water-soluble compounds, and incorporation of 14C in microbial biomass were more pronounced, but microbial biomass 14C declined more rapidly in litter than in soil. In short-term (14 day) incubations, mineralization of cellulose substrates generally corresponded with cellulase and xylanase activities in litter and soil. Pre-incubation with trace amounts of unlabeled holocellulose significantly increased the decomposition of 14C-labeled cellulose substrates and increased cellulase activity later in the experiment but did not affect xylanase activity. The sum of 14C02production, ~4C in microbial biomass, and a4C in watersoluble compounds is considered to be a sensitive parameter by which to measure cellulolytic activity in soil and litter samples in short-term incubations. Shorter periods than 14 days are preferable in assays using Acetobacter cellulose, because the decomposition of this substrate is more variable than that of holocellulose and plant cellulose.

Offprint requests to. S. Scheu.

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Introduction

The decomposition of cellulose in litter and soil is considered to provide a measure of the biological activity [2]. A variety of methods are used to determine the cellulolytic activity and cellulose decomposition in litter and soil, including the loss of strength and weight in cotton strips [9,10] and the activity of cellulases [13,19]. The aim of the present study was to investigate the usefulness of incubating 14C-labeled cellulose substrates as a sensitive in situ incubation method for analyzing decomposition processes. For this purpose, three different 14C-labeled substrates (holocellulose and plant cellulose, prepared from 14C-labeled beech (Fagus sylvatica) seedlings, and bacterial cellulose from Acetobacter xylinum) were incubated for 14 days in litter and soil samples which had been pre-incubated for different time periods with and without addition of trace amounts of unlabeled holocellulose. Changes in the conversion of labeled substrates to 14CO2, incorporation of 14C in microbial biomass, and the amount of labeled water-soluble compounds were monitored. The cellulose substrates were also incubated in litter and soil for 154 days and conversion to 14CO2, 14C incorporation in microbial biomass, and production of labeled water-soluble compounds were monitored. Materials and Methods

Litter and Soil In May 1990, samples of leaf litter from the litter layer (L horizon) and of soil (upper 3 cm of the mineral soil; An horizon) were taken from a beechwood growing on limestone in Northern Germany (G6ttinger Wald [20]). The soil is of the Rendzina type and rich in humus. After removing twigs and other non-leaf materials by hand, the leaf litter material [Corg content 50.6%, C/N ratio 37; determined by a Carlo Erba (Milano, Italy) elemental analyzer] was cut to pass a 10-mm screen, and the mineral soil (Corg content 8.9%, C/N ratio 14) passed through a 4-ram sieve. Samples were stored at 4°C in sealed polyethylene bags for 6 weeks.

14C-labeled Substrates Stems and twigs of beech seedlings which had been incubated in a plant growth chamber containing about 0.04% 14CO2 (during two subsequent vegetation periods) [7] were cut manually into small pieces and then ground to pass a l-ram screen. Water-soluble and lipid compounds were removed by washing three times with a methanol/chloroform/water solution (10:2:1) [4]. Then proteins and starch were extracted twice using pronase E from Streptomyces griseus and amyloglucosidase-rohalase from Aspergillus niger (Serva Company, Heidelberg, Germany). Holocellulose was prepared following Wise et al. [25] by incubating the pre-extracted wood material three times with NaC102 solution at 70°C for 1 h. The extracted material was cleared three times with water and freeze-dried. The specific activity of the holocellulose was 137 kBq g 1. Holocellulose from unlabeled beech twigs was prepared in the same way. 14C-labeled plant cellulose was prepared by separating hemicellulose from the ~4C-labeled holocellulose material using the procedure of Dickson [4]. A subsample of ~4C-labeled holocellulose was incubated in a 10% KOH solution at 30°C for 24 h (2x). After centrifugation and decantation, the material was cleared with cold ethanol-4M acetic acid (2×), ethanol (2x), water (3x) and then freeze-dried. The specific activity of this material was 122 kBq g-1. Acetobacter cellulose as thin films was prepared from A. xylinum cultures [8,12] fed with 14Clabeled glucose using the procedure of du Preez and Kistner [6]. Since Acetobacter cellulose is

Decomposition of 14C-labeled Cellulose

289

organized in simple ribbon like structures and not incorporated in a non-cellulosic matrix [23], unlabeled Acetobacter cellulose was added to 14C-labeled Acetobacter cellulose to obtain material of similar specific activity to the 14C-labeled holocellulose and 14C-labeled plant cellulose. The labeled and unlabeled materials were homogenized in water using a laboratory mixer.

Long-term Incubation of 14C-labeled Cellulose Substrates Litter (200% moisture by dry weight) and soil samples (60% moisture) equivalent to 12.5 and 50.0 g dry weight, respectively, were incubated in darkness at 15°C in glass jars of 1.5 and 0.5 1, respectively. To each of 12 litter and soil jars, 15 mg of 14C-labeled holocellulose plant cellulose, or Acetobacter cellulose was added and the materials thoroughly mixed with a spatula. Evolved 14CO2, trapped in alkali (1N KOH) in small vessels placed on the bottom of each jar, was determined by liquid scintillation counting of an aliquot after 7, 14, 28, 42, 56, 70, 84, 98, 119, and 140 days. Overall CO 2 produced in the jars was also determined titrimetrically after 7, 14, 21, 49, 77, 105, and 140 days [15]. Generally, the alkali was replaced at weekly intervals. Cumulative production of 14CO2 and total CO 2 were calculated assuming linearity between measurements. After 14, 49, 84, and 154 days, three jars from each treatment were destructively sampled to determine microbial biomass I4C and the amount of 14C-labeled water-soluble compounds (see below).

Short-term Incubation of 14C-labeled Cellulose Substrates Litter and soil were incubated in glass jars as described above (24 replicates each). Unlabeled holocellulose (15 mg) was added to 12 jars with soil and 12 jars with litter. The jars were incubated at 15°C in permanent darkness. After 14, 42, 70, and 126 days, three jars were destructively sampled and their contents devided into two portions of 1/3 (A) and 2/3 (B). In A materials, enzyme activities were determined (see below). B portions were divided into six aliquots. To two of these, 10 mg of 14C-labeled holoceIlulose, plant cellulose, or Acetobacter cellulose were added and the material then incubated in 0.5-1 glass jars in darkness at 15°C for 14 days. Evolved 14CO2 was determined as described above. Half of the jars containing soil or litter incubated with holocellulose, plant cellulose, or Acetobacter cellulose were then fumigated with chloroform prior to determining microbial biomass

I4c.

Microbial Biomass 14C Microbial biomass 14C was determined by the fumigation extraction method [21]. Litter and soil samples equivalent to about 1.3 and 5.5 g dry weight, respectively, were fumigated with chloroform (stabilized with 20 ppm 2-methyl-2-butene) for 24 h and subsequently extracted with 80 ml 0.5 N K2SO 4 solution on a rotary shaker for 30 min. Unfumigated samples were extracted in the same way. 14C in the extracts was determined by liquid scintillation counting. Microbial biomass 14C was calculated from the difference of 14C in fumigated and unfumigated samples using a k c of 0.45 [26]. In addition to microbial biomass 14C, the amount of water-soluble 14C was calculated from the amount of label in unfumigated 0.5 N K2SO4 extracts. Two parameters of a4C incorporation in microbial biomass were calculated in the short-term incubation experiment: (1) the amount of label incorporated in microbial biomass as percentages of the initial C content of labeled cellulose substrates (14Cmic)and (2) the assimilation efficiency of a4C in cellulose substrates (AEcs) calculated as: AEcs = 14Cmic X 100/(14Cmic 4- 14CO2-C) The amount of water-soluble a4C-labeled compounds was not incorporated in the calculation of the AE because it might be caused mainly by cell-free enzymes in litter and soil samples.

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Enzyme Activities Fresh soil samples equivalentto 1 g dry weight were extractedin 5 ml sodiumacetate-aceticacid buffer (0.5 M, pH 5.0) including 1 mg NaN 3 m1-1 to prevent microbial growth. Assays of endo-acting cellulase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) activities were performedin micro-plates (pH 5.0, 40°C, 15 min for L material, 120 min for Ah material) as describedby Wirth and Wolf [24]. One unit of enzyme activitywas calculated as absorbance × 100 x min-~.

Statistical Analysis Two-, three-, or four-wayANOVAs were used to analyzedata on cumulative 14CO2-Cevolution, 14C incorporationin microbialbiomass, amountof 14Cin water-solublecompounds, and enzymeactivities. The data were inspected for homogeneity of variances (Bartlett-Box F) and log transformed, if necessary, to approximatehomogeneityof variances prior to ANOVAs. Four-way,complete,randomized block ANOVA was used to analyze cumulative 14CO2-Cevolution and ~4C incorporation in microbial biomass in the short-termincubationexperimentwith labeled cellulose substrates. The data were blocked in the pre-incubationjar from which B materials were taken.

Results

Long-term Incubation of Cellulose Substrates 14C02 Evolution. The pattern of 14CO2-Cevolution from Acetobacter cellulose, holocellulose, and plant cellulose was very similar in litter and soil throughout the experiment, except during the first 14 days (Fig. 1). Mineralization rates were high during the first half of the experiment and then decreased progressively. During the first 7 days, 14CO2-C release from holocellulose exceeded that from Acetobacter cellulose, whereas after 14 days (soil) or 21 days (litter) 14CO2-C release from Acetobacter cellulose was considerably higher than that from holocellulose. A total of 98.2% of the variation in cumulative 14CO2 evolution could be explained by the treatments, with significant contributions from both STRATUM (litter and soil) (28.7%) and CELLULOSE (all types) (69.2%) and no significant interaction between the latter. In general, cumulative 14CO2-C evolution from Acetobacter cellulose exceeded that from holocellulose and plant cellulose by factors of 1.3 and 1.8, respectively. Mineralization of cellulose substrates was generally more pronounced in litter than in soil (Fig. 1). At the end of the experiment, cumulative ~4CO2-C evolution from cellulose materials incubated in litter exceeded that incubated in soil by a factor of 1.4. Total C02 Production by the Systems. Total CO2 production pooled for the three 14C-labeled cellulose substrates (there was no significant difference between these) in jars containing litter declined during the first 21 days and then remained almost constant (Fig. 2). In contrast, total CO 2 production in jars containing soil increased during the first 14 days and then decreased continuously until the end of the experiment. In general, total CO 2 production from litter considerably exceeded that from soil. A total of 22.0% and 4.0% of the initial C content of litter and soil, respectively, was mineralized during the experiment.

Decomposition of 14C-labeledCellulose

291

60 ¢-.

._o

~40

i

r

i

I

~

E

I

¢.-

0

40

80

120

160

Days Fig. 1. Cumulative14CO2-C evolutionfrom 14C-labeledholocellulose(I), plant cellulose (e), and Acetobacter cellulose (~,) (% of initial C content) incubatedin (a) litter and (b) soil (Ah layer) of a beechwoodon limestoneat 15°Cfor 140 days.

14Cin Microbial Biomass. In general, ]4C in microbial biomass decreased during the experiment, with TIME (at destructive samplings) the most important source of variation, but with significant contributions also from STRATUM and CELLULOSE (Table 1). Incorporation of 14C from cellulose substrates in microbial biomass was generally more pronounced in litter (overall mean of 14Cmic of 4.1%) than in soil (overall mean of 14Cmic of 2.7%) and in the order Acetobacter cellulose > holocellulose > plant cellulose (overall means of 3.9%, 3.2%, and 3.0%, respectively). Incorporation declined continuously from 6.9% at day 14 to 2.3 % at the end of the experiment in litter (Fig. 3), whereas it increased from 1.9% at day 14 to 5.2% at day 48 and then decreased to below detection level by the end of the experiment in soil. The decrease in 14C in microbial biomass from Acetobacter cellulose between 48 and 84 days was more pronounced than from holocellulose and plant cellulose, which was mainly caused by the strong decrease in label in microbial biomass of the mineral soil (Fig. 3). The incorporation of label from the three cellulose substrates was generally similar in litter and soil (no significant interaction of CELLULOSE and STRATUM), and the pattern of incorporation was similar at each of the sampling dates in litter and soil (no significant three-way interaction).

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"O "7 0.15

>

O tO

0.10

-.a 0.05

9

G O

I

I

40

I

/

I

80

I

I

120

160

Days Fig. 2.

Rates of total C O 2 - C production (% of initial C content per jar and day) by experimental jars containing litter (I) and soil (A h layer) (o) of a beechwood on limestone incubated at 15°C for 140 days.

Table 1. Three-way ANOVA with data on 14C-incorporation in microbial biomass from 14C-labeled cellulose substrates (as percentages of initial 14C content in cellulose substrates) and data on the amount of 14C in water-soluble compounds (as percentages of the amount of label remaining in the jars). Factors were TIME (destructive samplings at days 14, 48, 84 and 154), STRATUM (litter and soil), and CELLULOSE (holocellulose, plant cellulose, and Acetobacter cellulose). 14C in microbial biomassa

TIME STRATUM CELLULOSE TIME X STRATUM TIME X CELLULOSE STRATUM x CELLULOSE TIME x STRATUM x CELLULOSE Residual

14C

in water-soluble compounds a

SS b ( % )

F c,d

SS b ( % )

F c,d

39.9 7.0 1.6 28.4 6.8 1.4 3.9

63.0*** 37.2"* 4.7* 44.8*** 5.4*** 2.7ns 3.1ns

2.9 42.4 18.6 3.8 12.4 3.8 7.3

5.2** 230.9"** 50.8*** 7.0** 2.3*** 7.1"** 6.8***

10.1

8.8

a log

transformed data bSS = sum of squares CF = F-value arts = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

14C in Water-Soluble Compounds. Generally, more labeled water-soluble compounds were extracted from litter than from soil (overall means of 2.7% and 1.1%, respectively) with STRATUM accounting for most of the variation (Table 1). The amount of labeled water-soluble compounds differed between the cellulose substrates; generally, more 14C was extracted from Acetobacter cellulose (2.6%) than from holocellulose (1.7%) and plant cellulose (1.4%) (Fig. 4).

293

Decomposition of 14C-labeled Cellulose 10

8-

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o 0

55

i

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I

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40

80

120

Fig. 3,

160

Days

Incorporation of 14C in microbial biomass (as percentages of the initial ~4C in cellulose substrates) from a4C-labeled holocellulose, plant cellulose, and Acetobacter cellulose mixed in (a) the litter and (b) the mineral soil layer (Ah horizon) of a beechwood on limestone during 154 days of incubation (legend as in Fig. 1; for statistical analysis see Table 1).

Although water-soluble compounds from Acetobacter cellulose in total exceeded those from holocellulose and plant cellulose, at day 14 they were lower in litter and only slightly higher than those from holocellulose and plant cellulose in soil (Fig. 4).

Short-term Incubation of Cellulose Substrates 14C02 Evolution. Total release of 14CO2-Cfrom cellulose substrates mixed in litter markedly exceeded that of the substrates mixed in soil by a factor of 3.5 (Fig. 5) with STRATUM accounting for most of the variation (Table 2). Mineralization of the cellulose substrates was similar at day 0 and the first sampling period (overall means of 7.0 and 6.3% of initial C content 14-day -1, respectively) and then increased rapidly until the third sampling period (overall mean of 22.3 % of initial C content 14-day- 1) with the effect of TIME being highly significant. Generally, the mineralization rates of holocellulose and plant cellulose were similar (overall means of 10.4% and 9.2% of initial C content 14-day -1, respectively) and considerably lower than that of Acetobacter cellulose (overall mean of 12.7% of initial C content 14-day-1) although they were 2.5 times higher at the first sampling date (Fig. 5). The greater mineralization of Acetobacter cellulose was generally more pronounced in mineral soil than in litter. Pre-incubation with unlabeled holocellulose also strongly affected the mineralization of cellulose substrates (Fig. 5, Table 2). However, whereas mineralization

294

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Fig. 4. Changes in the amount of 14C-labeled water-soluble compounds (% of 14C remaining in the jars) derived from holocellulose, plant cellulose, and Acetobacter cellulose mixed in (a) litter and (b) soil (Ah layer) of a beechwood on limestone during 154 days of incubation (legend as in Fig. 1; for statistical analysis see Table 1).

in soil was increased by a factor of 1.8, in litter it was increased only slightly (Fig. 5). Pre-incubation with holocellulose generally affected the mineralization of the three cellulose substrates similarly [no significant interaction of PRE-HOLOCELLULOSE (pre-incubation with or without addition of unlabeled cellulose) and CELLULOSE] and was independent of the sampling dates (no significant interaction of PRE-HOLOCELLULOSE and TIME). However, as shown by significant three-way interactions, the effect of preincubation with holocellulose varied among the sampling dates in litter and soil (PRE-HOLOCELLULOSE × TIME x STRATUM) and differed among the three cellulose substrates (PRE-HOLOCELLULOSE x TIME x CELLULOSE; Table 2, Fig. 5).

14C in

Microbial Biomass. Incorporation of label in microbial biomass in litter markedly exceeded that in soil by a factor of 2.6, with the effect of STRATUM being highly significant (Table 2). In soil, incorporation of label was low at the first sampling period and increased strongly to the second sampling period (by a factor of 3.9). In litter, incorporation of label remained more constant (Fig. 6-1). Incorporation of label from the cellulose substrates was generally most effective from plant cellulose (overall mean of 8.1%) and considerably lower from Acetobacter cellulose and holocellulose (overall means of 6.5% and 5.9%, respectively). 14Cmic from holocellulose, plant cellulose, and Acetobacter cellulose varied strongly with time. ~4Cmic from Acetobacter cellulose incubated in soil increased dramatically from the first to the second sampling date, whereas 14Cmio from holocellulose and plant cellulose increased only moderately (Fig. 6-1).

Decomposition of 14C-labeled Cellulose

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40

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Pre-incubation (days) Evolution of 14CO2-C from 14C-labeled holocellulose, plant cellulose, and Acetobacter cellulose during 14 days of incubation (% of initial 14Ccontent of labeled cellulose substrates). Labeled cellulose substrates were mixed in (a) litter and (b) soil (Ah horizon) of a beechwood on limestone. Litter and soil had been pre-incubated for 14, 42, 70, and 126 days without holocellnlose (k~]), plant cellulose ([~l), and Acetobacter cellulose ([~), and with the addition of unlabeled holocellulose (~'q), plant cellulose ([~), and Acetobacter cellulose ([7-A)before labeled cellulose substrates were added. Evolution of 14CO2-C from cellulose substrates mixed in litter and soil at day 0 is given as control (for statistical analysis see Table 2). Fig. 5.

Pre-incubation with unlabeled holocellulose caused a significant increase in 14Cmic (Fig. 6-I, Table 2), the effect being more pronounced in soil than in litter (increase in 14Cmic by 55% and 17%, respectively). In litter, the effect of the pre-incubation with holocellulose increased rapidly until the second sampling and decreased later in the experiment, whereas in soil the effect of the pre-incubation was most pronounced at the last sampling period. The treatments also strongly affected the assimilation efficiency of ~4C in cellulose substrates (AEcs) (Table 2). The overall mean of AEcs was 32.4% and 39.8% in litter and soil, respectively, but varied with time (Fig. 6-II). In general, AEcs was

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Table 2. Four-way randomized complete block ANOVA with data on 14CO2-Crelease (% of initial C content) from 14C-labeled cellulose substrates, 14C incorporation in microbial biomass (% of initial C in cellulose substrates) and the assimilation efficiency of C from cellulose substrates (%) during the incubation of labeled cellulose substrates mixed in pre-incubated litter and soil of a beechwood on limestone for 14 days. Factors were TIME (pre-incubation for 14, 42, 70 and 126 days), STRATUM (litter and soil), CELLULOSE (holocellulose, plant cellulose, and Acetobacter cellulose) and PREHOLOCELLULOSE (pre-incubation with and without unlabeled holocellulose); the data were blocked in the pre-incubation jar. 14CO2-C releasea

TIME STRATUM CELLULOSE PRE-HOLOCELLULOSE TIME x STRATUM TIME x CELLULOSE TIME x PREHOLOCELLULOSE STRATUM x CELLULOSE STRATUM x PREHOLOCELLULOSE CELLULOSE x PREHOLOCELLULOSE TIME × STRATUM × CELLULOSE TIME × STRATUM × PRE-HOLOCELLULOSE TIME x CELLULOSE x PRE-HOLOCELLULOSE STRATUM x CELLULOSE x PRE-HOLOCELLULOSE TIME x STRATUM x CELLULOSE x PREHOLOCELLULOSE PRE-INCUBATION JAR Residual

14C in microbial biomass a

Assimilation efficiencya

SSb(%)

. F c,~

SSb(%)

Fc.d

SSb(%)

Fc,~

26.4 41.4 1.9 2.6 3.3 12.6 0.1

667.9*** 3145.7"** 72.3*** 194.2"** 84.4*** 159.9"** 2.3ns

11.2 36.6 2.9 3.8 15.9 9.6 0.6

66.5*** 652.0*** 25.8*** 67.3*** 94.8*** 28.4*** 3.7*

42.0 7.2 8.9 0.1 13.6 3.3 0.5

186.8"** 96.5*** 59.7*** 1.7ns 60.7*** 7.4*** 2.3ns

1.4 2.4

52.0*** 185.7"**

1.9 0.6

16.6"** 11.2"**

1.7 2.1

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0.1

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0.5

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4.9

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a log transformed data bSS = sum of squares CF = F-value a n s = P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001

at a m a x i m u m at the first s a m p l i n g date and d e c r e a s e d until the third s a m p l i n g date~ but in soil it r e m a i n e d m o r e c o n s t a n t t h a n in litter, w h e r e it d r o p p e d d r a m a t i c a l l y . T h e A E w a s m a r k e d l y g r e a t e r in plant c e l l u l o s e ( o v e r a l l m e a n o f 4 1 % ) than in h o l o c e l l u l o s e and A c e t o b a c t e r c e l l u l o s e ( o v e r a l l m e a n s o f 3 6 % and 3 2 % , r e s p e c t i v e l y ) (Fig. 6-II, T a b l e 2), A l t h o u g h the o v e r a l l m e a n s o f AEcs in s a m p l e s prei n c u b a t e d and not p r e - i n c u b a t e d w i t h u n l a b e l e d h o l o c e l l u l o s e w e r e a l m o s t identi-

Decomposition of 14C-labeled Cellulose

o~60

(la)

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126

Pre-incubation (days)

(lib)

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126

Pre-incubation (days)

Fig. 6. Incorporationof ~4C in microbial biomass from 14C-labeledholocellulose,plant cellulose, and Acetobacter cellulose mixed in (a) litter and (b) soil (Ah horizon) of a beechwoodon limestone during 14 days of incubation; (I) percentagesof the initial 14C in cellulosesubstrates and (II) assimilation efficiency of C from cellulose substrates. Litter and soil were pre-incubated without and with unlabeled holocellulosefor different periods of time (legend as in Fig 5; for statistical analysis see Table 2).

cal, in litter the pre-incubation caused an increase in AEcs from 31% to 35%, whereas in soil it caused a decrease from 42% to 38%.

Enzyme Activities. The activity of cellulase and xylanase varied between sampling dates in litter and soil (Fig. 7). Activities of both enzymes in litter considerably exceeded that in soil, with STRATUM being highly significant (Table 3). In contrast to cellulase, the activity of xylanase was strongly affected by TIME and decreased strongly in both litter and soil later in the experiment (Fig. 7). The decrease was more pronounced in litter than in soil. The effect of pre-incubation with unlabeled trace amounts of holocellulose varied also with time (Table 3) and pre-inculbation caused an increase in cellulase activity later in the experiment, especially in soil. Discussion In preliminary experiments, the release of 14C02-C from labeled holocellulose, plant cellulose, and Acetobacter cellulose incubated in litter and soil of the study site increased almost linearly within the first days of incubation [Scheu, unpublished, 14CO2-C released during the first 2 days and days 5-7 were strongly correlated (r 2 = 0.99; n = 18) and 14CO2-C release during days 0-7 and days 8-14

298

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Pre-incubation (days)

Pre-incubation (days)

Fig. 7. Enzyme activities in (a) litter and (b) soil (A h horizon) of a beechwood on limestone; (I) cellulase, (II) xylanase. Litter and soil were pre-incubated without (El) and with holocellulose ([]) (for statistical analysis see Table 3).

Table 3. Three-way ANOVA with data on cellulase and xylanase activities (units) in litter and soil (A h horizon) of a beechwood on limestone. Factors were TIME (pre-incubation for 14, 42, 70 and 126 days), STRATUM (litter and soil), and PRE-HOLOCELLULOSE (pre-incubation without and with addition of unlabeled holocellulose). Cellulase activity a

TIME STRATUM PRE-HOLOCELLULOSE TIME × STRATUM TIME × PRE-HOLOCELLULOSE STRATUM × PRE-HOLOCELLULOSE TIME × STRATUM × PRE-HOLOCELLULOSE Residual '~log transformed data bSS = sum of squares CF = F-value arts = P > 0.05; *P < 0.05; P < 0.001

Xylanase activity a

SS b (%)

F C'a

SS b (%)

F c'd

0.5 95.4 0.1 0.1 0.9 0.2 0.4

2.0ns 1255.9"** 1.9ns 0.4ns 4.0* 2.3ns 1.8ns

9.1 78.8 0.1 5.9 0.6 0.6 0.2

16.6"** 431.9*** 0.7ns 10.8"** 0.Ins 0.3ns 0.3ns

2.4

5.8

Decomposition of 14C-labeled Cellulose

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were also correlated strongly ( r 2 = 0.97; n = 24)]. In the present study, an incubation period of 14 days was preferred to shorter incubation periods to ensure that the incorporation of label in microbial biomass was detectable; previous findings that conversion of cellulose C to microbial tissue C is slow, particularly in non-preincubated samples, were confirmed. Only 0.9-2.2% of C of holocellulose, plant cellulose, and Acetobacter cellulose mixed in mineral soil from the study site (not pre-incubated at 15°C) could be recovered in microbial tissue after 14 days (Fig. 3). In general, both the short-term incubation with labeled cellulose substrates and the cellulase and xylanase enzyme assays gave similar evidence of cellulolytic activity in litter and soil samples. Overall means of cellulase and xylanase activity in litter exceeded that in soil by factors of 62 and 48, respectively, and the overall mean rate of C mineralization of the three labeled cellulose substrates mixed in litter exceeded that in soil by a factor of 3.5. However, in contrast to the cellulase activity, the conversion of cellulose substrates to 14CO2 depended strongly on the duration of pre-incubation. In soil, C mineralization of labeled cellulose substrates increased strongly from day 14 to day 42, whereas in litter the increase was most pronounced between day 42 and day 70 (Fig. 5); these increases were not accompanied by increased extractable cellulases. Except between day 14 and 42 in litter material, the xylanase activity in litter and soil decreased with pre-incubation. In contrast, a decrease in C mineralization of cellulose substrates was detected only at the last sampling date. A strong increase followed by a decrease in cellulase activity and loss in cellulose compounds during laboratory incubations of litter was observed by Linkins et al. [14], who postulated a shift from mainly cellulose-decomposing fungi to microorganisms able to attack phenolic compounds and cellulose material simultaneously. The control of litter decomposition by the lignin fraction is stressed frequently [1,5]. Possibly due to higher incubation temperatures, the drop in cellulose decomposition was more pronounced in the study of Linkins et al. [14] than in the present experiment, where only 22% and 4% of the initial C content of the litter and soil material, respectively, was converted to CO2-C during the incubation period of 140 days. Except during the first 21 days, CO2-C evolution from litter remained on a very constant level, whereas CO2-C evolution from soil decreased continuously later in the experiment (Fig. 2), indicating that depletion of utilizable organic matter occurred only in soil. Incorporation of label in microbial biomass followed a similar pattern to that of mineralization of the cellulose substrates. In comparison to soil, 14Cmic was increased in litter by a factor of 2.6. In soil 14Cmic and the mineralization of the cellulose substrates among sampling dates varied more than in litter and increased strongly from the first to the second sampling period (Fig. 5, Fig. 6-I). In contrast to C mineralization and 14Cmio, the assimilation efficiency of label from cellulose substrates ( A E J was similar in litter and soil (overall mean of 32% and 40%, respectively), reached a maximum at the first sampling date (overall mean of 50%), and varied more intensively in litter than in soil (Fig. 6-II). Similar AE~s in litter and soil indicate similar efficiencies of the litter and soil microflora to convert C in cellulose substrates to microbial tissue C. The decrease in the efficiency of conversion of cellulose C to microbial tissue C in the litter microflora after the third sampling date suggests that the microorganisms dominat-

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ing in later stages in litter decomposition are less efficient in resource allocation for growth, due to expending more energy in the exploitation of more refractory C resources. The substrate conversion efficiency was greatest for plant cellulose (overall mean of 41%) and considerably lower for holocellulose and Acetobacter cellulose (overall means of 36% and 32%, respectively), indicating adaptation of the litter and soil microflora to the use of C resources similar to plant cellulose. The release of 14C02-C differed among the three labeled cellulose substrates. In the long-term incubation experiment, microbial conversion of C to 14COz-C in Acetobacter cellulose exceeded that of C in holocellulose and plant cellulose. C mineralization of the cellulose substrates in litter exceeded that in soil considerably. Hence, the lower efficiency of soil microorganisms in comparison to the litter microflora to mineralize cellulose substrates observed in short-term incubation experiments was also valid for the long-term incubations. More intensive mineralization of holocellulose in comparison to plant cellulose was expected because the hemicellulose fraction in holocellulose is known to be less resistant to microbial attack than cellulose [18]. Acetobacter cellulose consists of uniformly arranged cellulose microfibrils in ribbon-like layers [23] lacking the complex arrangement of cellulose (and hemicellulose) microfibrils in plant cell walls. In addition, the degree of polymerization is lower in bacterial cellulose than in plant cellulose [ 17]. Due to its uniformity, bacterial cellulose is considered to be an ideal model substrate for degradation studies [23] and has been used to analyze the cellulase enzyme system of several fungi [3]. However, presumably due to the uniformity of cellulose microfibril arrangement and a lower degree of polymerization, Acetobacter cellulose is decomposed more rapidly than plant cellulose materials. This was also observed in the short-term incubation experiment where the conversion of C from holocellulose and plant cellulose was similar and considerably lower than that from Acetobacter cellulose. In addition, in comparison to holocellulose and plant cellulose, mineralization of Acetobacter cellulose varied more intensively with pre-incubation (Fig. 5). More pronounced conversion of C in cellulose substrates to 14C02-C in litter in the long-term experiment was accompanied by a greater amount of label incorporated in the litter microflora in comparison to microorganisms of the mineral soil. In litter, 14Cmic decreased continuously during the experiment, whereas in soil it increased for 48 (holocellulose and Acetobacter cellulose) or 84 days (plant cellulose). Despite the fact that the incorporation of label in microbial biomass differed among the three cellulose substrates and was generally4in the order Acetobacter cellulose > holocellulose > plant cellulose, changes in 1 C incorporation from each of the cellulose substrates were very similar during the long-term incubation (Fig. 3). As with the conversion of C from labeled cellulose substrates to 14CO2-C and 14Cmic, the production of water-soluble compounds from the three labeled cellulose substrates was generally in the order Acetobacter cellulose > holocellulose > plant cellulose, and the production of water-soluble compounds in litter considerably exceeded that in soil. The amount of water-soluble compounds from plant cellulose in litter and soil remained constant throughout the experiment, whereas that from Acetobacter cellulose varied considerably and that from holocellulose declined slowly. The total cellulose decomposition ability of microorganisms in litter and soil samples can be determined by adding together 14CO2-C, 14Cmic, and

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the amount of labeled water-soluble compounds. However, it is presumably necessary to add greater amounts of labeled cellulose substrates to ensure the accessibility of the substrates to all of the cellulolytically active microorganisms. One of the main advantages of using radiotracers in decomposition experiments is that even very small amounts of labeled substrates are sufficient to follow the decomposition pattern in the sample. The addition of very small amounts of labeled substances is considered to cause little disturbance of the microbial environment, and the carbon turnover of samples can be studied under conditions closely resembling the undisturbed system. However, even the addition of small amounts of tracer material may affect decomposition processes in the sample. It is well known that the decomposition of labeled substrates added to soil samples increases with the amount of substrates added [11,16]. Pre-incubation with trace amounts of unlabeled holocellulose in the present study showed that even amounts equivalent to the addition of 0.12% and 0.34% of litter and soil C, respectively, altered the ability of the microflora to decompose cellulose substrates. Pre-incubation with unlabeled trace amounts of holocellulose generally caused an increased in the conversion of C in labeled cellulose substrates to 14CQ-C and in 14Cmic, whereas AEos was affected inconsistently and was different in litter and soil. Despite the fact that the pre-incubation with holocellulose altered the shortterm turnover of cellulose substrates strongly, no effect on xylanase activity could be directed and an increase in cellulase activity could be detected only later in the experiment. Presumably, the short-term conversion of labeled cellulose substrates to 14CO2-C and 14Cmic are more sensitive parameters for detecting the cellulose degrading ability of the microflora. The effect of pre-incubation with unlabeled holocellulose on the conversion of C from labeled cellulose substrates to 14CO2 and 14Cmic was different in litter and soil. In litter, mineralization of labeled cellulose substrates was almost unaffected by pre-incubation with unlabeled holocellulose, whereas in soil, the pre-incubation caused a strong increase in the conversion of cellulose C to ~4CO2-C. The more pronounced effect of the addition of trace amounts of holocellulose on the soil microflora may be related to a more pronounced activation of cellulose decomposing microorganisms. Presumably, a considerable amount of the soil microflora was in a less active phase [22] and was partly activated during the pre-incubation with holocellulose. The present results show that the short-term decomposition of cellulose substrates is related to cellulase and xylanase activity in soil and litter samples. The conversion of C in cellulose substrates to 14CO2-C and the incorporation of cellulose C in microbial biomass during short-term incubations are shown to be sensitive parameters for measuring the cellulose decomposition ability in soil and litter samples. The sum of 14CO2-C, 14Cmic, and the amount of 14C in water-soluble compounds presumably is the most powerful parameter for measuring the cellulolytic activity in litter and soil samples in a short-term incubation assay. Each of the cellulose substrates used was a sensitive indicator of cellulose decomposition ability. A short-term incubation assay based on process analysis of cellulose decomposition is assumed to be a useful tool to characterize the cellulolytic activity in litter and soil. Of the three cellulose substrates, the decomposition of holocellulose and plant cellulose were more similar and differed from Acetobacter cellulose.

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Presumably, using Acetobacter cellulose, shorter incubation periods should be chosen to characterize the cellulose decomposition ability of soil and litter samples, because Acetobacter cellulose can be attacked more rapidly and the decomposition varies more intensively than that of holocellulose and plant cellulose. To ensure high sensitivity in assays lasting only a few days, cellulose substrates with higher specific activity than those used in this study are preferable. In contrast to cellulose materials of plant origin, Acetobacter cellulose of higher specific activity can be generated easily. Acknowledgments. We are grateful to D. Hartwig for technical assistance in generating labeled cellulose substrates. Suggestions by Professor G.A. Wolf and two anonymous reviewers improved the manuscript considerably. The study was partly supported by the Bundesministerium fiir Forschung und Technologie.

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