Biology and Fertility
Biol Fertil Soils (1987) 5:164-170
Activity, origins and location of cellulases in a silt loam soil
C,E A. Hope* and R.G. Burns
Biological Laboratory, University of Kent, Canterbury, Kent CT2 7N J, UK
Summary. "Cellulase" activity in a silt loam soil was
assayed and characterised using a microcrystalline cel-
lulose substrate (Avicel). Activity was maximal be-
tween p H 5.3 and p H 6.0. A 64% loss in activity was
observed on air-drying the soil. However, the residual
activity was stable to storage at 40 °C for 7 days and
was resistant to the action of added protease. The
component endoglucanase and ~-D-glucosidase ac-
tivities in field-moist and air-dried soil were also as-
sayed. The proportion of the soil microbial popula-
tion able to utilise cellulose was investigated and the
persistence of two free (soluble) cellulase preparations
of microbial origin was determined following their
addition to soil. A rapid decline in the endoglucanase
activity of a S t r e p t o m y c e s sp. cellulase preparation
was observed while 30% of the original activity of a
T r i c h o d e r m a viride cellulase preparation could still be
detected after 20 days. From the data obtained in this
study it appears that the major portion of the ~-D-
glucosidase activity is bound to and protected by the
soil colloids. By contrast, the major portion of the
exo- and endoglucanase activity appears to be "free"
in the soil solution, attached to the outer surfaces of
cellulolytic microorganisms or associated in enzyme
substrate complexes. The low residual activity mea-
sured in air-dried soil may owe its stability to an asso-
ciation with soil colloids or with recalcitrant cellulosic
material present in soil.
Key words: Soil cellulase - Enzyme characterisation
- ~-D-Glucosidase - Endo-
T r i c h o d e r m a viride -
glucanase
* Present address: Department of Microbiology, University of Sur-
rey, Guildford, Surrey, GU2 5XH, UK
Offprint requests to: R. G. Burns
°f S o i l S
© Springer-Verlag1987
The exact mechanism whereby microorganisms and
enzymes decompose cellulose is still the subject of
some controversy (Burns 1982a) and the variety of
names applied to the cellulolytic enzymes and their ac-
tivites have added to the confusion (Lee and Fan
1980). In this paper we have confined our discussion
to the activities of the enzymes endo-l,4-13-D-
glucanase (EC 3.2.1.4), exo-l,4-[3-D-glucanase (cello-
biohydrolase - EC 3.2.1.91) and [3-D-glucosidase
(EC 3.2.1.21) and we have used the term "cellulase"
to refer to the combined action of these enzymes on
Avicel - a purified depolymerized alpha cellulose
derived from fibrous plants.
A variety of methods and substrates have been
used for measuring cellulolytic activity in soils, e.g.
field measurements made on cotton strips or film
(Latter and Howson 1977), measurement of carbon
dioxide evolved from labelled (Ibister et al. 1980) and
unlabelled (Sato 1981) cellulose, and release of reduc-
ing sugars from carboxymethyl cellulose (Pancholy
and Rice 1973) and cellulose powder (Benefield 1971).
Most of these studies have been concerned with deter-
mining the overall cellulase activity of the soil for
comparative reasons. More recently, carboxymethyl
cellulose and cellulose powder have been used to
study the properties and location of the cellulase com-
plex in a tomato field soil (Hayano 1986).
In this study we have attempted to assay and to
characterise (in terms of pH-activity profile, thermo-
stability, etc.) the total cellulase as well as the com-
ponent endoglucanase and [3-D-glucosidase activities
in a silt loam soil. This, together with a study of the
cellulolytic microbial population present in the soil
permits us to provide a detailed description of these
enzyme activities and to speculate as to their location
in the bulk soil (Burns 1982b).

Materials and methods
Soil A silt loam soil (Hamble Series: Soil Survey Record No. 14)
with the following characteristics was used: p H 6.4, sand 9o70, silt
72%, clay 19%, organic matter 6% (determined by ignition of
oven-dried soil); CEC 14.8 cmol kg -1 (Lethbridge and Burns
1976). The level of residual carbohydrate present in the soil at the
time of collection was 7.8 mg (g soil)-1 (measured as glucose equiv-
alents following hydrolysis with 1.5 M H z S O 4 according to Brink et
al. (1960). Enzyme activity was measured both in freshly collected
and air-dried soil. Where it was impossible to perform assays
immediately, fresh soil was stored for short periods (<48 h) in
sealed plastic containers at 4 °C. Alternatively, air-dried soil (72 h,
22 °C) was hand crumbled and sieved through 2-mm mesh prior to
storage in dark-glass bottles at room temperature (22 o+ 3 °C).
Viable counts o f soil microorganisms. Triplicate samples of flesh
soil (1 g) were suspended in 100 ml sterile buffer (7 g NazHPO4, 3 g
KHzPO4,5 g NaC1, 1-1). The flasks were shaken on a rotary shaker
(150 rpm) for 1 h at 30°C to dislodge microorganisms from the soil
particles and a tenfold dilution series in buffer prepared. Three 0.1-
ml aliquots of three consecutive dilutions were spread onto agar
plates and the plates incubated at 25 °C for 7 days. Total bacterial
counts were made on nutrient agar plates whilst cellulolytic bacteria
were counted on plates containing Avicel PH105 (Honeywill and
Stein, Wallington, UK). The Avicel plates were prepared by over-
laying a defined minimal medium, pH 7.5 (Berg et ai. 1972a) con-
taining 1% w/v purified agar (Oxoid), with 10 ml of the same
medium to which 1% w/v Avicel had been added. The total num:
ber of fungal colonies was counted on malt extract agar plates and
cellulolytic fungi on Avicel plates prepared in the same manner as
the bacterial plates but using a minimal medium, p H 4.6 (Stern-
berg, 1976). In order to prevent the growth of any bacterial colonies
on the fungai plates, filter-sterilized streptomycin sulphate (400 ~tg
ml - l ) was added to the partially cooled (ca. 45°C) autoclaved
media.
Cellulase assay. Combined exoglucanase (EC 3.2.1.91), endoglu-
canase (EC 3.2.1.4), and 13-D-glucosidase (EC 3.2.1.21) activity -
hereafter collectively referred to as "cellulase" - was measured by
placing 1 g soil in a 25 ml Erlenmeyer flask and adding 5 ml 0.1 M
acetate buffer, pH 5.5, containing 0.2070 (w/v) sodium azide, as a
bacteriostatic agent. Washed Avicel (0.5 g) was added, the flask
sealed and incubated at 40°C for 16 h in a shaking water bath. In
the control Avicel was added at the end of the incubation period
and the reaction stopped by centrifugation (2500xg, 10 min). Each
supernatant was assayed in duplicate for reducing sugars and/or
glucose so that the levels o f product glucose and cellobiose could be
calculated. Reducing sugars were determined using the Nelson-
Somogyi copper reduction method (Spiro 1966) and glucose by a
modified glucose oxidase method (Pycraft and Howarth 1980). In
both methods the absorbance readings were related to glucose con-
centration by means of a standard curve (0.05 - 0.4 mM) which was
prepared each day. In the Nelson-Somogyi assay, the time that the
samples were kept in the 100 °C water bath prior to the addition of
the arsenomolybdate reagent was set at 20 rain since this was the
boiling time required for a standard solution (0.25 mM) of cel-
lobiose to give the same absorbance reading as a standard glucose
solution (0.25 mM) assayed under identical conditions. Three
replica treatments and three controls were used for each assay. A
glucose standard curve was constructed at each pH since the redox
potential of the sugars is pH dependent.
In assays where humic matter was extracted into the super-
natant and interferred with the colorimetric assay (e.g. when buf-
165
fers other than sodium acetate were used to investigate pH-activity
profiles) the humic matter was precipitated by the sequential addi-
tions of 100 Ixl 1.2 M-lead acetate [(CH3COO)2Pb3H20 ] and 100 ~tl
1.2 M-potassium oxalate [(COOK)2H20 ] .
The stability of glucose produced during the course of the assay
was examined by performing an "assay" in which Avicel was re-
placed by glucose (0.05 - 0.5 re_M). Reducing sugars and glucose in
the supernatants were measured immediately after addition, after
16h incubation at 40°C, and following lead acetate/potassium
oxalate treatment. Glucose added directly to soil was recovered
quantitatively (100% + 9%) after 16 h incubation, indicating that
under the conditions of the assay (in the presence of 0.2% (w/v)
NaN 3 to prevent microbial proliferation) glucose was not utilized
by soil microorganisms, adsorbed to soil colloid, or oxidised to any
significant extent by indigenous soil glucose isomerase (Ross 1974).
However, following precipitation of extracted humic material, only
80% ___5% of the added glucose could be detected. This 20°70 + 5%
loss was probably due to coflocculation of glucose with humic acids
and a compensation factor (xl.25) was introduced for assays in
which this treatment was used.
Endoglucanase. Endoglucanase activity (EC 3.2.1.4) was measured
using a soil to buffer ratio of 1 • 5 and 1%0 (w/v) CMC 7L (Hercules
Inc., Wilmington, Del. USA) as the substrate. Samples were in-
cubated unders shaking conditions for 16h at 40°C. Reducing
sugars in the supernatant following centrifugation (2500xg,
10 min) were determined using dinitrosalicylic acid reagent - DNS
(Miller et al. 1960).
fl-D-Glucosidase. 13-D-Glucosidase activity was measured by mix-
ing 0.2 g air-dried soil with 1.5 ml 0.1 M-acetate buffer, pH 5.5,
and 1.0ml 25 mM-p-nitrophenyl-13-D-glucopyranoside (pNPG).
Controls without p N P G we also prepared. After incubation (4 h,
307C), 0.5 ml of 0.5 M CaC12 was added to each bottle and I ml
p N P G was added to the controls. The reaction was terminated by
addition of 2 ml 0.5 M Tris, pH 10.5, and the mixtures centrifuged
(2500x g, 10 min). The supernatants were then diluted as appropri-
ate and the absorbance at 400 nm determined. The absorbance
readings were related to p N P concentrations by means of a stan-
dard curve.
Characterization o f enzyme activities. The thermal stability of
the soil cellulase activity was determined by bringing air-dried soil
to ca. 85%0 W H C [0.5 ml 1-120 containing 0.2% (w/v) NaN 3
(g soil)- 1] and storing the samples in 25 ml Erlenmeyer flasks at
25 °C or 40°C for up to 14 days.
The resistance of the soil cellulase activity to proteolytic attack
by a nonspecific protease (Sigma type VI) was determined. Com-
parison was made with a Trichoclerma viride commericai cellulase
(BDH) subjected to the same treatment. Soil samples (1 g) at 85%
water-holding capacity (0.5 ml 0.1 M Tris/HC1 buffer, pH 7.0,
containing 0.2% NaN 3 and 3 mg ml-1 protease) were maintained at
25 °C. Because the added protease was found to be gradually in-
activated in the soil samples, two additional amendments were
made (1.5 mg protease after 24 h and 67 h) in order to maintain a
high level of diffusible (free) protease activity. The presence of dif-
fusible protease activity was confirmed, during and at the comple-
tion of the experiment, using the method of Burns et al. (1972).
Trichoderma viride cellulase (1 mg m1-1 , prepared in 0.1M-
Tris/HCl pH 7.0, containing 0.2 % NaN 3 ) was subjected to 0.1 and
1.0mg m1-1 protease (equivalent to ca. 16% and 138% of the
calculated average diffusible (free) protease activity measured in
the treated soil samples). Cellulase activity in the protease-treated
and untreated soils was assayed in the usual manner except that
166

4.5 ml 0.1 M-acetate buffer was used. The m e t h o d used to assay the Table 1. Viable counts of microorganisms in Hamble soil [numbers
protease-treated and untreated Trichoderma cellulase preparations (g dry wt. soil)- 1 and standard deviation (o n_ 1)]
was the same as that used for soil except that 1 g soil was replaced
with 1 ml enzyme preparation and the a m o u n t of buffer reduced Medium Counts (cfu x l 0 - 5)
from 5 ml to 4 ml.
The persistence (resistance to degradation by native soil pro- Bacterial Nutrient agar 378 _+51
teolytic enyzmes or due to adsorption without inactivation to soil Avicel/Berg M M 70 _+16
colloids) of the endoglucanase c o m p o n e n t of Trichoderma viride Fungal Malt extract agar 8.1 _+4.1
commercial cellulase and a cell-free cellulase preparation from a Avicel/Sternberg M M 7.2 ___3.9
Streptomyces sp. soil isolate (Hope and Burns 1985) was deter-
mined by adding aliquots (0.2 ml) of cellulase preparation (1 mg
m1-1 in 0.1M-acetate buffer, p H 5.5, containing 0.2% (w/v)
N a N 3) to 0.4 g air-dried soil. The enzyme-treated soil and an un- 100
treated control were incubated at 25 °C and assayed for endoglu-
canase activity at intervals using the usual endoglucanase assay ex-
8O
cept that the incubation period was reduced to 30 min and the DNS
reagent added directly to the soil slurry.
60

Results and discussion -~ 40

Viable counts o f soil microorgan•ms 2o

From a comparison of the total bacterial counts on
nutrient agar and the cellulolytic counts on Avicel 0
,

0-02 0"05
i

0"10
i

0"15
i

plates (Table 1), 7 × 106 or ca. 20% of the soil bacteria
were able to utilize Avicel as their sole carbon source.
However, only a small proportion (ca. 0.2%) of the
cellulolytic bacteria produced clearing zones on the
Avicel plates (indicative of extensive hydrolysis and
unequivocal evidence of their use of the substrate). A
large number (ca. 80%) of the cellulolytic bacteria
were actinomycetes. Reports in the literature on the
cellulase system of the actinomycetes (Crawford and
Sutherland 1979; Hagerdal et al. 1980; Ishaque and
Kluepfel 1980) suggest that these organisms possess a
cellulase system more akin to that of the fungi than
the bacteria in that their cellulases are secreted to the
surrounding medium. One factor limiting the growth
of bacteria other than the actinomycetes on solid
media containing Avicel is that, in order to utilize this
particular substrate, microorganisms may need to
align themselves along the cellulose fibril. Evidence
for this view comes from electron microscope studies
of bacterial configuration (Berg et al. 1972b) and ex-
periments which indicate that many bacterial species
retain some of their cellulases at the outer membrane
rather than secreting them to the surrounding medium
(Ramasamy and Verachtert 1980). Therefore bacteria
may have difficulty in growing on Avicel plates
because the microcrystalline cellulose particles are in-
corporated in the agar and many are not directly ac-
cessible as carbon and energy sources.
Fungal counts on Avicel plates were not signifi-
cantly different from those on malt extract agar plates
(Table 1). Unlike the bacterial Avicel plates, where a
small number of distict zones of clearing were
observed, clearing zones were not seen at all on the
fungal Avicel plates. However, with many of the
0.20
Avicet Concentration (g rnl"q)
Fig. 1. Effect of substrate (Avicel)concentration on cellulaseactivi-
ty [nmol reducing sugars as glucose equivalents (g dry wt. soil)-1
h -1] in field moist soil (26O7owater content) and soil: buffer ratios
of o; 1:2; and A; 1:5
fungal colonies the dense sporulating mycelium made
it impossible to see any clearing zones. Furthermore,
a comparable study made using a Trichoderma viride
isolated from soil indicated that the radial growth rate
of the fungal colony on CMC agar plates was almost
twice that of the rate of diffusion of endoglucanase,
thereby masking any visual signs of hydrolysis (Hope
and Burns 1985). Therefore, even if the advancing
fungal hyphae were producing endoglucanase one
would not expect to see a zone of clearing advancing
ahead of the colony perimeter. This illustrates the im-
portance, when screening fungi for cellulase produc-
tion by the zone method, of either making an allow-
ance for the rate of radial growth or else limiting
colony size by incorporating an agent such as rose
bengal into the medium (Montenecourt and Eveleigh
1977).
Soil cellulase activity
When soil was incubated with the substrate Avicel
(0.1 g m l - t) the rate of release of reducing sugars was
constant for the first 24 h after which it declined.
Figure 1 shows the effect of different substrate con-
centrations (0.002 - 0.2 g ml - i) on cellulase activity at
25 °C for two different soil to buffer ratios. Substrate
saturation kinetics were not observed. Increasing the
amount of Avicel relative to the amount of soil either

by increasing the Avicel concentration in the buffer or
by increasing the buffer to soil ratio increased the
amount of reducing sugars that were produced. The
most likely explanation for this is that the Avicel,
while being chemically homogeneous, is not physical-
ly homogeneous and some regions, e.g. those surfaces
of the elementary fibrils where "strain-distorted tilt
and twist" occur, will be more susceptible to rapid
hydrolysis than others (Rowland 1975). Several
kinetic models of varying complexity have been
proposed to explain the hydrolysis of insoluble
cellulose by cellulase enzymes.
However, an unified model has not yet emerged.
Our system is further complicated by the fact that
some of the enzymes whose activity we are measuring
are likely to be soil bound or otherwise immobilised
on microbial cell walls (Burns 1982b). As the sub-
strate is also insoluble it is likely that steric hindrance
and restricted diffusion of the interactants would
make saturation of all the active sites impossible. It is
unlikely that cellobiose is causing end product inhibi-
tion of enzyme activity, for while this phenomenon
has been reported for purified cellulases it is only
operative at cellobiose concentrations of greater than
30 m M (Reese 1963), i.e. ca. two orders of magnitude
higher than that observed in the soil assays. An
incubation period of 16 h and a substrate concentra-
tion of 0.1 g m l - i was chosen for the routine assay so
that the rate of product formation would be constant
for the duration of the assay and the results could be
expressed in standard units.
Field moist soil (26.3o/0 moisture) contained only
a very low level of extractable reducing sugars
( < 50 nmol g - a) and the level of these extracted into
the buffer did not increase during the incubation
period of the assay. By contrast, when air-dried soil
was assayed 6.25 nmol g - l h - 1 reducing sugars were
produced during the course of the assay in the absence
of added substrate. This was attributed to the air-dry-
ing process increasing the accessibility of endogenous
substrate. The carbohydrate content of the soil was
43.3 ~mol glucose equivalents g - 1.
p H activity profile
The relationship of cellulase activity to pH for air-
dried soil is shown in Fig. 2. The activity is maximal
between 5.25 and 6.0. The pH optima for fungal cel-
lulases is in the range 4.0 - 5.5 while bacterial (includ-
ing the actinomycete) cellulases have pH optima in the
range 5 . 6 - 7 . 0 . This suggests that a wide range of
microbial types are contributing to the overall activity
in our soil. However, direct comparisons of the prop-
erties of soil enzymes with those from microbial
sources must be made with caution for two reasons.
167
~0
30
~ 20
10
~O
s:0 s:o 70
pH
Fig. 2. Effect of pH on soil cellulase activity [nmol reducing sugars
(solid lines) or glucose (broken lines), expressed as glucose equiva-
lents (g dry wt. soil) -1 h - l ] . A, A; sodium acetate buffer; O, © ;
sodium acid maleate buffer; IN; Tris/maleate buffer
Firstly the pH of the soil-buffer slurries in which these
assays are performed is almost certainly not the same
as that at the soil/solution interface, where much of
the enzyme/substrate interaction is probably occur-
ring. In fact the pH at the soil colloid surface may be
considerably more acid than that of the bulk solution
due to the accumulation of H ÷ ions within the diffuse
double layer (McLaren and Skujins 1968). Secondly,
the activity and the pH profile of the soil cellulases are
probably a composite of a group of enzymes from
microbial as well as plant origins. Fungi have been re-
ported as being the principal producers of extracel-
lular cellulases (Enari and Markkanen 1977) and since
the true p H at which the cellulase is most active
(taking into account the interfacial phenomena) is
likely to be below 5.6, the cellulase activity that we
have measured is probably of fungal origin. A similar
conclusion was reached by Hayano (1986) for the
cellulase activity measured in a tomato field soil.
Nevertheless, there are many reports in the literature
on the cellulolytic activities of soil actinomycetes
(Crawford and Sutherland 1979; Williams and Robin-
son 1981) and the possibility that members of this
group are contributing to the pool of cellulase activity
measured in our soil should not be ignored.
The measurement of glucose produced during the
16-h assay demonstrated that this sugar accounted for
65% - 80o70 of the total reducing sugars. Although the
apparent pH optima of the [3-D-glucosidase com-
ponent of cellulase in this soil was 5.2 - 5.5, the high
level of glucose produced was predicted since the ac-
tivity of the [3-D-glucosidase enzyme in air-dried soil
is an order of magnitude higher than the "cellulase"
activity we were measuring and it is likely that any
free cellobiose would be rapidly hydrolysed to glucose
even at suboptimal pH values. The remaining
(20O/o-35O/o) reducing sugars are likely to be corn-
168
posed of a small amount of cellobiose and the higher
cellooligosaccharides that are produced during cellu-
lolysis.
Thermal stability and resistance
to proteolytic attack
No significant change in cellulase activity was ob-
served following storage of air-dried soil moistened to
85% W H C at 40°C for 14 days. Thermal stability is a
characteristic often associated with soil-bound
enzymes (Pettit et al. 1977; Nannipieri et al. 1982);
also cellulase-humic complexes have been shown to
have increased thermal stability when compared with
free (soluble) cellulase (Sarkar 1986).
The effect of added protease on cellulase activity
in soil compared with that of a Trichoderma viride
commercial cellulase preparation (BDH) is shown in
Fig. 3. Virtually all T. viride commercial cellulase ac-
tivity was lost by 24 h after treatment with 1.0 mg
ml-1 protease. The soil cellulase activity in contrast
was resistant to attack by protease (3 mg m l - 1). The
resistance o f soil cellulase to proteolytic attack is not
surprising considering the high levels of protease ac-
tivity and proteolytic microorganisms found in
Hamble soil. Only resistant enzymes will survive to be
assayed. Soils collected at different times of the year
(March, June and October) and air-dried had similar
activities towards Avicel (34.3, 38.2 and 39.8 nmol
reducing sugars [as glucose equivalents (g dry wt.
soil)- 1 h - 1], suggesting that this portion of the total
activity is constant regardless of season.
Endoglucanase activity
Reducing sugars were released at a linear rate for the
first 30 h following the addition of CMC to either
field-moist soil or air-dried Hamble soil. In a 16-h as-
say the activity in field-moist soil was 490 nmol reduc-
ing sugars as glucose equivalents (g dry wt. soil) - t h - 1
and the activity in air-dried soil was l l 0 n m o l g - t
h - t . These activities were not characterised further.
fl-D-Glucosidase activity
The ~-D-glucosidase activity was largely unaffected
(10070- 15% loss) by air-drying the soil. The activity
in air-dried soil was measured as 0.88 ~tmol p N P
(g soil) -1 h -1 with a pH optimum of 5 . 2 - 5 . 5 and a
temperature optimum of 60°C. This activity was re-
sistant to added protease, indicating that it was bound
to and protected by the soil colloids. This was later
confirmed when about 10% of the original stable ac-
tivity was detected in filter-sterilised humic extracts
(Pettit et al. 1977) and was shown to have properties
similar to that of the whole soil activity.
1 2 0 ~
oo\
0 24 48 72
Time(h)
Fig. 3. Effect of protease on free (soluble) and soil cellulase activity
(expressed as a percentage of the untreated control), o ;
soil+protease (3 mg ml-1); ©; commerical cellulase+protease
(0.1 mg m l - 1); []; commercial cellulase + protease (1.0 mg m l - 1)
Location o f cellulase activity in bulk soil
The cellulase activity measured in field-moist Hamble
soil [ca. 110 nmol reducing sugars as glucose equiva-
lents (g dry wt.)- 1 h - 1 at 40 °C] is comparable to that
reported by Beneficial (1971) for an agricultural soil
(3200nmol glucose g-a 4 8 h - 1 at 50°C) and that
reported by Ross and Speir (1979) for two silt loam
New Zealand soils [25.2 and 50.4nmol reducing
sugars as glucose equivalents (g dry wt.)-1 h - I at
30 °C]. Assuming a linear relationship with time and
activity and a Q10 effect of approximately 2, the
activity reported by Benefield is 31, those by Ross and
Speir are 50.4 and 100.8 respectively, and that ob-
served here is 110.8 units.
In contrast to the [3-D-glucosidase activity, a 64%
decrease in cellulase activity was observed on air-dry-
ing. A similar decrease ( 3 6 % - 80%) in activity fol-
lowing air-drying was reported by Speir and Ross
(1981) for a range of soils assayed for cellulase activi-
ty with cellulose powder. A decline of ca. 50% in
[~-l,3-glucanase activity was observed by Lethbridge
et al. (1978), and they suggested that the stress of air-
drying denatured much of the extracellular unbound
and hence unprotected enzyme. The significant per-
centage of the cellulase activity measured in field-
moist soil is either associated with soil microorgan-
isms which are not resistant to air-drying or is free in
the soil solution. The low residual cellulase activity as
compared with, for example the residual [~-l,3-glu-
canase activity (ca. 400 nmol reducing sugars as glu-
cose equivalents g-1 h-1 _ Lethbridge et al. 1978),

80
6O
o
20 . ~ , . ,
0 mo 200 3oo 46o s60
Time (h)
Fig. 4. Persistence in soil of Trichoderma viride commerical cel-
lulase preparation (O) and a cellulase preparation from a Strepto-
myces sp. (A). Endoglucanase activity expressedas a percentage of
the "soil-free" preparation stored at 25 °C
combined with the inherent stability and variety of
free extracellular endoglucanase (Goksoyr et al.
1975), makes deductions concerning the location of
these enzymes in soil difficult.
The results of experiments in which the persistance
in soil of the endoglucanase component of T. viride
commercial cellulase and cellulase from a Strepto-
myces sp. isolate were investigated are shown in Fig.
4. A rapid decline in the endoglucanase activity of the
Streptomycete sp. cellulase preparation was observed.
By contrast, although the endoglucanase activity of
the T. viride cellulase preparation showed an initial
decline following its addition to soil, nearly 30% of
the initial activity could nonetheless be detected after
20 days. This clearly illustrates the ability of some,
but not all, endoglucanases to withstand inactivation
due to adsorption, denaturation and degradation.
Drowzdowicz (1971) measured both endoglucanase
and the combined exo- and endoglucanase activity of
an Aspergillus niger commercial cellulase following
its addition to soil. He found that while endo-
glucanase activity declined slowly over a 40-day
period the combined exo- and endoglucanase activity
of the added preparation disappeared with 24 h. In
studies where the in vitro stability of the different
components of the cellulase complex have been in-
vestigated (e.g. Hagerdal et al. 1980), it appears that
the activity of the endoglucanase component has
greater stability than that of the combined exo- and
endoglucanase activity. The comparative instability
of the combined exo- and endoglucanase activity most
likely reflects the instability of the exoglucanase com-
ponent. However, measurements of purified exo-
glucanase are needed before this can be confirmed. It
is impossible to assay specifically for exoglucanase
activity in soil because of the synergistic action of the
169
enzymes involved in the cellulase complex. [Purified
exoglucanases are generally identified by their lack of
activity against C M C and their ability, when com-
bined with endoglucanase, to bring about the exten-
sive hydrolysis of crystalline cellulose. In addition,
release of cellobiose units f r o m H3PO4-swollen cellu-
lose has on occasion been used as a measure of exo-
glucanase activity (Wood et al. 1980).]
The results obtained in this study lead us to
propose that the m a j o r portion of the exo- and endo-
glucanase activity in field-moist soil is "free" in the
soil solution, attached to the outer surfaces of cellulo-
lytic microorganisms or associated in enzyme sub-
strate complexes. The high counts of cellulolytic fungi
and actinomycetes lends credence to this view and
while it is recognised that m a n y of the colony-forming
units may have originated f r o m resting propagules
and m a y therefore not be representative of the active
microbial biomass present in soil, they nonetheless are
indicative of a large potential microbial cellulolytic
population. The low residual activity measured in air-
dried soil may owe its stability to an association with
soil colloids or with recalcitrant cellulosic material
present in the soil. The hydrolysis of insoluble sub-
strates by enzymes immobilised by association with
soil colloids poses certain conceptual difficulties in
terms of substrate-enzyme interaction. However, the
colloidal nature and hence mobility of the enzyme
support (clay, humates) in the soil aqueous phase go
some way towards overcoming these difficulties.
Acknowledgments. We thank the Agricultural and Food Research
Council for supporting this research and R. J. Geraghty for
technical assistance.
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Received September 29, 1986