- University Of Nigeria Nsukka

University of Nigeria
Research Publications
METUGE, Jonathan ALUNGE
Amylase And Cellulase Activities In Forest-Savanna
Mosaic Soils In Nsukka.
Agriculture
Date
Department
Title
PG/M.SC/86/4417
Faculty
Author
+++
Soil Science
Signature
November, 1988
CERTIFICATION
T h i s i s t o c e r t i f y t h a t t h i s work was done by Metuge,
Jonathan Alunge i n t h e Department o f S o i l S c i e n c e , Faculty of
A g r i c u l t u r e , U n i v e r s i t y o f N i g e r i a , Nsukka.
D r . N . N . Agbim
S e n i o r Ldcturer,
Supervisor.
P r o f e s s o r W.O. Enwezor
Head o f Department
DEDICATION
T h i s t h e s i s i s d e s e r v e d l y d e d i c a t e d t o Nyango Epote-Wang
Metuge who s t r u g g l e d t o bring m e up i n t h e absence o f h e r l a t e
husband.
ACKNOWLEDGEMENTS
deep appreciation goes to Dr. N.N.
Agbim, my project
supervisor for reviewing the final manuscript, making corrections
where necessary.
I am greatly
W.O.
He was always available when I needed him.
indebted to the Head of Department, Professor
Enwezor and the entire members of the academic staff for
offering me an opportunity into the field of soil science.
particularly thankful to Professor WOO. Enwezor, Dr. N.N.
I am
Agbim
and Dr. I. Unaeba-Oparah for their inspiring lectures in soil
science.
I am grateful to Dr. I.U!
Obi for his useful advice on the
statistical methods adopted to analyse the soil enzyme data.
I also
vish to thank Mr. C-Jo Onyirioha for making readily available some
of the equipment used in this work and for some technical advice.
My kind
affection goes to my brother, Dr. W. Metuge for his
contribution to my education.
thank my friends, A.A.
For their spirit of comradeship I
Dickson, C-A. Igwe and D-0. Asawalam.
too to Dr. SON- Alrwe and P.T.
Thanks
Etuge for their concern and moral support.
Finally, I am grateful to the Cameroon government for the
financial assistance.
Metuge J- Alunge.
CONTENTS
Page
i
Title Page
ii
Certification
iii
Dedication
iv
Acknovledgement
Contents
List of Tables
List of Figures
Abbreviations
Abstract
INTRODUCTION
**
L1TERA'NJR.E REVIEW
o
MATERIALS AND METHODS
**
R E S n T S AND DISCUSSION
* .
CONCwSIONS
**
REFERENCES
**
*
L I S T OF TABLES
Page
TABLE
Mean values of the physical and chemical properties
of grassland soil (0 - 15 cm depth)
..
Mean values of the physical and chemical properties
of soil under shrubs (0 15 cm depth)
-
..
Mean values of the physical and chemical properties
of forest soil (0 - 15 cm depth)
..
Mean values of the physical and chemical properties
of forest-shrub-grassland mosaic soils (0 - 15 cm
depth)
..
..
Mean amylase activity in soils under different
vegetation covers
..
..
Monthly amylase activity in soils under three
different vegetation covers
..
Mean cellulase activity in soils under different
vegetation covers
..
..
Monthly cellulase activity in soil under three
different vegetation covers
..
Percentages of different organic components in
leaf litter collected from the floors of different
vegetation covers
..
..
Correlation coefficients (r) for climate and soil
factors and carbohydrase activities i n soils under
different vegetation covers
..
Multiple correlation coefficients (R) for
carbohydrase activities, soil moisture and organic
matter levels in the three vegetation types
LIST OF FIGURES
Page
FIGURE
1,
Seasonal variations in amylase activity in
forest-shrub-grassland mosaic soils with
corresponding monthly rainfall data
o o
2,
Seasonal variations in cellulase activity in
forest-shrub-grassland mosaic soi 1s with
corresponding monthly rainfall data
o
ABBREV I ATTON
E,C,
=
Enzyme classification
ABSTRACT
Amylase and cellulase activities were determined in surface
soils collected at monthly intervals over a one year period (~eb.
'87 - ~an,'88)from
three diverse vegetation types
-
forest,
shrubland and grassland, occurring within the same location,
Soil
amylase and cellulase activities were generally highest in grassland,
intermediate in shrubland and lowest in forest throughout the year
of study, in contrast to the trend in organic matter which was
highest in forest, intennediate in shrubland and lowest in grassland
soil.
During the rainy season, when soils remained moist, soil
enzyme activities increased, but' at the end of the rainy season, as
soils dried out their activities were again reduced in soils under
all the three vegetation types.
Monthly variations in amylase
activity were higher in forest than in grassland and shrubland soils,
while for cellulase activity such variations were slightly higher
in grassland soil than in forest or shrubland soil.
Significant negative correlations were found between the
activities of the enzymes under study and soil pH or organic matter
content,
Analysis of fallen plant litter from the canopy of the
three vegetation types showed that leaf litter from the grassland
vegetation had a higher cellulose to lignin and starch to lignin
ratios than did litter from shrubs and forest vegetation cover.
The
type of vegetation and thus the type of organic matter that returned
naturally to the soil seemed to be the chief determinant of the
activity gradients of amylase and cellulase in soils under field
conditions.
Significant negative correlations were also observed
between the soil enzyme activities and such other soil properties
as C/N ratio, per cent base saturation ('$I BS), and total exchangeable
bases (TED), in contrast to the significant positive correlations
observed with rainfall, soil moisture and available phosphorus.
possible mechanisms
involved in these associations are discussed,
although they cannot be resolved from available data.
Although soil moisture seemed to account for most of the
variations in soil amylase and cellulase activities, this factor
b
was moderated by the type of org-anicmatter present under each
vegetation type.
The
Also, most other relationships between enzyme
activity and other soil factors appeared to be predicated by the
type of litter fall under each vegetation.
INTROWCTION
Forest-savanna mosaic is a v e g e t a t i o n c o v e r i n which t h e humid
t r o p i c a l r a i n f o r e s t and a d e r i v e d savanna e x i s t i n t h e same l o c a t i o n .
I n t h e s e mosaic areas, t h e f o r e s t s a r e i n most c a s e s t h i c k w h i l e t h e
a d j a c e n t s o i l c o v e r i s predominantly g r a s s w i t h a few s c a t t e r e d young
trees,
The f o r e s t and t h e savanna grow on t h e same p a r e n t material
-
false-bedded s a n d s t o n e o r u n c o n s o l i d a t e d sedimentary materials ( J u o ,
1981; Akamigbo and Asadu, 1983; Unamba-Oparah,
1987b) and r e c e i v e t h e
sane amount o f r a i n f a l l and s u n s h i n e i n t e n s i t y , t h a t i s , have t h e
same t y p e o f c l i m a t e ,
t o be homogeneous
t r o p i c a l climate.
-a
Thus n a t u r a l l y one should e x p e c t t h e v e g e t a t i o n
b
t h i c k t r o p i c a l r a i n f o r e ~ t ~ b e c a u sof
e t h e humid
The savanna p o r t i o n i s r e f e r r e d t o a s d e r i v e d
savanna implying i t s anthropogenous d e r i v a t i o n from a n i n i t i a l f o r e s t
vegetation,
H i s t o r y shows t h a t t h e s e a r e a s were o r i g i n a l l y covered
w i t h f o r e s t but i n d i g e n o u s c u l t u r a l p r a c t i c e s l e d t o t h e c u t t i n g down
of t h e trees f o r farmland,
Constant burning of t h e farmland gave
rise t o a f o r e s t - s a v a n n a t y p e o f v e g e t a t i o n ,
T h i s t y p e of v e g e t a t i o n
i s caarmonly observed i n most p a r t s of s o u t h - e a s t e m N i g e r i a
( I g b o m r i k e , 19751, p a r t i c u l a r l y i n Imo and Anambra s t a t e s , where
w i t h i n a f e w m e t e r s , o n e c a n p a s s from a c l o s e d f o r e s t t o a n open
grassland.
Depending on t h e cropping i n t e n s i t y , t h e v e g e t a t i o n c o v e r
might form a f o r e s t - s h r u b l a n d - g r a s s l a n d mosaic, where t h e s h r u b l a n d
r e p r e s e n t s a r e l a t i v e l y less i n t e n s e l y cropped savanna a r e a , e x i s t i n g
as a mixture o f t a l l g r a s s e s and a few s c a t t e r e d young t r e e s ,
In
t h i s s t u d y t h e r e f o r e , savanna r e f e r s t o both g r a s s l a n d and s h r u b l a n d
vegetations all derived from a forest vegetation due to human
activity.
As the pressure on the land increases due to overpopula-
tion, the derived savanna gradually encroaches into the humid tropical
rainforest of these areas.
Various studies (Ojanuga, 1980; Osakwe, 1981; Unamba-Oparah,
1987b3 have been carried out to assess the chemical and physical
characteristics of forest-savanna mosaic soils.
These studies give
an indication of how human activity could affect soil physical
and chemical properties and the susceptibility of the derived
b
savanna areas to soil erosion as these continue to be cropped.
However,
there is no literature on the biochemical characteristics of the
forest-savanna mosaic soils.
Ross (1966), suggested that whereas
seasonal or small local differences in vegetation or land management
may have little effect on the physical or chemical properties of the
soil as a whole, they could markedly influence its biochemical
behaviour.
One of the characteristic features of derived savanna
soils where most of the farmlands occur is their relatively low levels
of organic matter content (Unarnba-Oparah, 1982).
It is therefore
necessary to improve the organic matter content of these soils
characterized by low activity clays in order to increase their
potentials for food production (Juo, 1981; Agboola and Fagbenro,
1985; Unegba-Oparah, 1987a).
Soil organic matter supplies most of
the cation exchange capacity (CEC) of acid sandy soils, improves
the uater-holding and buffering capacities of sandy soils, reduces
-3phosphorus f i x a t i o n by s e s q u i o x i d e s , s e r v e s as a s t o r e - h o u s e
s o i l s t r u c t u r e and s t a b i l i z e s t h e s o i l
p l a n t n u t r i e n t s , improves
a g a i n s t e r o s i o n (Greenland a n d D a r t , 1972; A l l i s o n ,
Oparahand E m e w r ,
for
1978).
1973; Unamba-
Before t a k i n g r a t i o n a l s t e p s i n any
d i r e c t i o n t o improve t h e o r g a n i c m a t t e r s t a t u s i n d e r i v e d s a v a n n a
s o i l s , i t is necessary t o s t u d y t h e enzymatic a c t i v i t i e s of s o i l
p o l y s a c c h a r i d a s e s that m e d i a t e h y d r o l y s i s o f p l a n t m a t e r i a l s w i t h i n
t h e s o i l environment.
Most s t u d i e s on l e a f d e c o m p o s i t i o n o f West
African p l a n t s ( ~ e n &
n ~
a,,
19h9; Nye,
1961; Agbim, 1987) have been
done by u s i n g r e s p i r a t i o n o r l i t t e r bag t e c h n i q u e s , w i t h o u t due
b
c o n ~ i d e r a t i o nf o r t h e h y d r o l y t i c enzymes t h a t almos t e x c l u s i v e l y
mediate t h e decmposi t i o n process.
Amylase a n d c e l l u l a s e a r e enzymes i n v o l v e d i n t h e h y d r o l y s i s o f
some o f t h e most a b u n d a n t o r g a n i c compounds i n n a t u r e
cellulose respectively,
3t a l , ,
-
s t a r c h and
They a r e t r u e e x t r a c e l l u l a r enzymes ( K i s s
1975) a n d e x i s t i n t h e s o i l a s a c c u m u l a t e d a b i o n t i c
enzymes ( S k u j i n s , 1976) where t h e y are assumed t o t a k e p a r t i n t h e
hydrol*ic
d e p o l y m e r i z a t i o n o f p o l y s a c c h a r i d e s a s p a r t o f t h e rnine-
r a l i z a t i o n of s o i l o r g a n i c m a t t e r ( K i s s e t al.,
1975).
Sources o f
t h e a c c m u l a t e d enzymes are p r i m a r i l y m i c r o b i a l c e l l s ( ~ o f f m a n n , 1963;
C l a r k a n d S t o n e , 19651, a l t h o u g h some no d o u b t , o r i g i n a t e from p l a n t
r o o t s (Chang a n d B a n d u r s k i ,
1964; I l i e v a n d Kimenov, 1972; Ross,
1476: Hayano, 1985) a n d s o i l a n i m a l s (Ladd, 1978).
By d e f i n i t i o n ,
a c c m u l a t e d p o l y s a c c h a r i d a s e s a r e r e g a r d e d a s enzymes p r e s e n t a n d
a c t i v e i n a s o i l i n d e p e n d e n t o f immediate m i c r o b i a l p r o l i f e r a t i o n
-tt(Kiss&
&.,
1978).
Polysaccharidases decompose plant litter, hence
play an important role in the cycling of carbon in the soil and
hence the perpetuation of life bn our planet.
Although the activities of amylase and cellulase enzymes have
been reported in several soils (Drobnik, 1955; Markus, 1955; Galstyan,
1x5;Ross, 1 x 6 ; Benefield, 1971; Drozdowicz, 1971; Kong et al.,
1971; Pancholy and Rice, 1973ap; Kiss et al., 1978; Hayano, 1986;
Kanazawa and Miyashita, 1987), they have not been studied in Nigerian
soils.
Ross (1965; 1966), and Pancholy and Rice (1973a), showed
that amylase and cellulase activities vary with season and type of
vegetation cover.
However, such studies were either carried out by
incubating soils in the laboratory with different plant materials or
were perfonned on soils under vegetation types occurring in widely
different locations, whereby soil variations arising from differences
in parent materials could greatly influence the measured enzyme
activities.
In order to assess the effect of type of plant materials
added to the soil, on amylase and cellulase activities, this study was
carried out on soils collected from the field in which forest, shrubland and grassland exist in the same location forming a forest-savanna
mosaic vegetation cover.
It is hoped that studies of the activity
gradients of polysaccharidases like amylase and cellulase in these
soils would lead to a better understanding of some of the most judicious
ways of improving organic matter levels in our acidic sandy soils,
in terms of the type, amount and time of organic matter placement
-5in cultivated soils.
They may also indicate the relative rates of
organic matter decomposition and carbon cycling in soils under
different vegetation covers.
-6 LITERATURE REVIEW
1.
SOIL ENZYMES
N u t r i e n t c y c l i n g i n s o i l s i n v o l v e s biochemical, chemical
and physicochemical r e a c t i o n s , w i t h t h e biochemical p r o c e s s e s
b e i n g m e d i a t e d by m i c r o o r g a n i s m s , p l a n t r o o t s , a n d s o i l a n i m a l s
( T a b a t a b a i , 1982).
A l l b i o c h e m i c a l r e a c t i o n s are c a t a l y s e d by
enzymes, which are p r o t e i n s produced by l i v i n g c e l l s .
Enzymes
are c a t a l y s t s , that i s , t h e y are s u b s t a n c e s which w i t h o u t underg o i n g permanent a l t e r a t i o n c a u s e c h e m i c a l r e a c t i o n s t o p r o c e e d
a t f a s t e r rates.
Like a l l c a t a l y s t s , t h e y
b
(i)
(ii)
(iii)
(iv)
are
effective in
very small concentrations
s p e e d up a r e a c t i o n u n t i l it r e a c h e s e q u i l i b r i u m
are unchanged by t h e r e a c t i o n , a n d
exhibit s p e c i f i c i t y f o r certain limited reactions.
During t h e c o u r s e o f t h e r e a c t i o n , t h e c o m b i n a t i o n o f t h e
enzyme w i t h i t s s u b s t r a t e l o w e r s t h e a c t i v a t i o n e n e r g y o f t h e
l a t t e r so that c h a n g e s t h a t o t h e r w i s e might o c c u r o n l y a t
e l e v a t e d t e m p e r a t u r e s c a n p r o c e e d u n d e r normal p h y s i o l o g i c a l
conditions.
The f o r m a t i o n o f t h e t r a n s i e n t complex o f enzyme E
w i t h s u b s t r a t e S o c c u r s a t a l o c a t i o n i n t h e enzyme m o l e c u l e
t e n n e d t h e a c t i v e c e n t r e ( ~ e h n i n ~ e r1982).
,
The c o n f i g u r a t i o n
of t h e a c t i v e c e n t r e r e g u l a t e s t h e s u b s t r a t e s p e c i f i c i t y of t h e
enzyme.
The overall r e a c t i o n r e s u l t i n g i n t h e f o r m a t i o n o f
p r o d u c t s P can be summarized:
where K
1'
K
2
and K
3
are t h e r a t e c o n s t a n t s o f t h e i n d i v i d u a l r e a c t i o n s
a n d ES i s t h e t r a n s i e n t e n z y m e - s u b s t r a t e complex.
The rate o f a n
enzyme c a t a l y z e d r e a c t i o n i s d e t e r m i n e d by t h e rate a t which ES
d i s s o c i a t e s i n t o E + P.
Enzymes are d e n a t u r e d by e l e v a t e d t e m p e r a t u r e s a n d e x t r e m e pH
values.
P a r t o f t h e p h y s i o l o g i c a l a c t i v i t y o f many s o i l o r g a n i s m s i s
t o release enzymes i n t o t h e s o i l .
T h e s e enzymes c a t a l y s e b i o l o g i c a l
t r a n s f o n n i i t i o n s i n t h e s o i l , a n d t h e d e g r a d a t i o n p r o d u c t s become
a v a i l a b l e as n u t r i e n t s f o r t h e organisms.
Any n u t r i e n t s which a r e
n o t u t i l i z e d f o r c e l l c o n s t r u c t i o n are r e l e a s e d f o r subsequent u s e
by p l a n t s a n d i n d e e d o t h e r m i c r o o r g a n i s m s .
I t can be a s s u m e d 2 t r i o r i
that some o f t h e s e enzymes r e m a i n i n t h e s o i l i n a n a c t i v e s t a t e
o u t s i d e t h e l i v i n g c e l l s t h a t p r o d u c e d them ( S k u j i n s , 1 9 6 7 ) .
Soil
t h e r e f o r e a p p e a r s t o b e s i m i l a r t o a l i v i n g s u b s t a n c e ; a s m a l l lump
o f i t d i f f e r s from a n y n o n l i v i n g n a t u r a l body i n i t s a b i l i t y t o c a r r y
on i n n u m e r a b l e c a t a l y t i c r e a c t i o n s c h a r a c t e r i s t i c o f a l i v i n g t i s s u e ;
t h a t is, under a p p r o p r i a t e c o n d i t i o n s i t i s capable of b i o l o g i c a l
metabolism s i m i l a r t o t h a t o f l i v i n g organisms ( ~ k u j i n s ,1967).
This
rn~tabolisrneffedalbv i t s a c t i v e population ( b i l l i o n s of bacteria, fungi,
a l g a e , a c t i n o m y c e t e s , p r o t o z o a a n d o t h e r s o i l f a u n a ) is m e d i a t e d
a l m o s t e x c l u s i v e l y t h r o u g h t h e e x t r a c e l l u l a r enzymes t h e y produce.
Thus t h e most e s s e n t i a l i n d e x o f b i o l o g i c a l a c t i v i t y i n s o i l i s i t s
e n z y m e t i c a c t i v i t y , hence a s t u d y o f t h e l a t t e r may p r o v i d e a n
o b j e c t i v e view o f p r o c e s s e s t a k i n g p l a c e i n s o i l (Kuprevich a n d
Shcherbakova, 1971).
The t o t a l enzyme c a p a c i t y o f a s o i l c o n s i s t s o f enzymes s t i l l
w i t h i n t h e s o i l o r g a n i s m s ( i n t r a - o r e n d o c e l l u l a r enzymes) and t h o s e
s e c r e t e d i n t o t h e s o i l medium ( e x t r a c e l l u l a r enzymes o r exoenzymes).
I n t r a c e l l u l a r enzymes a r e o x i d a t i v e a n d e n e r g y y i e l d i n g w h i l e e x t r a c e l l u l a r enzymes are mainly h y d r o l y t i c .
Of s p e c i a l i n t e r e s t t o s o i l
b
s c i e n t i s t s are t h e e x t r a c e l l u l a r enzymes.
s o i l f o r d e c a d e s ( S k u j i n s a n d McLaren,
They can p e r s i s t i n t h e
1968) a n d are n o t i n f l u e n c e d
by f l u c t u a t i o n s of t h e o r g a n i s m s t h a t produced them.
They are
c h a r a c t e r i s t i c f o r e a c h s o i l t y p e a n d c o u l d be u s e d f o r meaningful
a s s e s s m e n t o f s o i l s ( ~ k u j i n s , 1967:
1976).
Some m i c r o b i o l o g i s t s r e g a r d a n enzyme a s e x t r a c e l l u l a r once
i t has p a s s e d t h r o u g h t h e plasma membrane o f t h e organism t h a t
produced i t (Glenn, 1976).
Others (Pollock,
1962) have r e s t r i c t e d
t h e e x t r a c e l l u l a r term t o t h o s e enzymes found s o l e l y i n t h e s u p e r n a t a n t o r s o i l medium a n d produced d u r i n g normal c e l l growth ( e x c l u d i n g
lysis).
The e x p r e s s i o n ! ? o i l
e x t r a c e l l u l a r enzyme'lhas come t o b e
a c c e p t e d as a p p l y i n g e s s e n t i a l l y t o t h a t f r a c t i o n which i s s e p a r a t e d
b o t h s p a t i a l l y a n d p r o b a b l y t e m p o r a l l y from i t s c e l l u l a r o r i g i n
( ~ u r n s , 1978).
"abiontic"
S k u j i n s (19761, h a s s u g g e s t e d a d o p t i n g t h e a d j e c t i v e
t o d e s c r i b e s o i l exoenzymes.
True e x t r a c e l l u l a r o r a b i o n t i c
enzymes are those actively secreted into the soil environment.
However, a proportion of the soil catalytic component is caused by
enzyme leakage from viable cells and rupture of moribund cells.
The extracellular enzymes are adsorbed on surfaces of colloidal soil
particles and are in some way covalently bound to inorganic and
organic macromolecular components (McLaren and Packer, 1970).
Soil
therefore, can be looked on as a system of humus and minerals
containing both imobilized enzymes and occluded microbial cells
( ~ c ~ e r e n1975).
,
Present research in soil knzymology has several facets
(Skujins, 1978).
One seeks basic understanding of the activity
characteristics of free enzymes in the heterogenous soil matrix and
their stabilization mechanisms associated with soil particulate
matter.
Another is defining their involvement in soil organic matter
turnover and in plant nutrition.
These efforts may indicate whether
the results of soil enzyme research could be used to judge soil
fertility.
Other pertinent questions concern the interactions of
abiontic enzymes with agrochernicals, their use in forensic science
(Thornton and McIaren, 1975) and the relevance of soil enzymology to
planetary exploration for detection of extraterrestial life ( ~ u r n s ,
1978).
1.1
Origin and Range of Enzymes in Soil
Enzymes in soil originate from animal, plant and microbial
sources (Ladd, 1978).
Early studies indicate that enzymes are deposited
- 10i n s o i l by m i c r o f l o r a , m i c r o f a u n a , a n d p l a n t d e b r i s .
Later s t u d i e s
( R o s s , 1976) w i t h s t e r i l e r o o t s y s t e m s a l s o i m p l i c a t e r o o t e x u d a t e s a n d
sloughed-off
While m i c r o o r g a n i s m s are t h o u g h t t o con-
root c e l l s .
t r i b u t e l a r g e l y t o t h e a c c u m u l a t e d a c t i v i t y ( ~ o f f m a n n , 19631, e x c r e t i o n
by p l a n t r o o t s a n d s o i l f a u n a may be a m a j o r s o u r c e i n some i n s t a n c e s
a n d may a l s o i n d i r e c t l y s t i m u l a t e enzyme p r o d u c t i o n .
Thus t h r e e
types o f s o u r c e s o f f r e e enzymes i n s o i l a p p a r e n t l y e x i s t .
(a)
P r o l i f e r a t i n g microorganisms and dying microorganisms
( f r o m which t h e enzymes a r e r e l e a s e d b e c a u s e o f
c h a n g i n g p e r m e a b i l i t y + o f t h e i r c e l . 1 membranes).
(b)
S o i l animals.
(c)
P l a n t r o o t s and p l a n t residues.
The t r u l y e x t r a c e l l u l a r enzymes, m o s t l y h y d r o l a s e s ( c a r b o x y lesterase, arylesterase,
l i p a s e , phosphatase, n u c l e a s e s and nucleo-
t i d a s e s , phytase, a r y l s u l p h a t a s e , amylase, c e l l u l a s e , lichenase and
larminarinase, inulase, xylanase, dextranase, levanase, pectinase,
a-
and @ - g l u c o s i d a s e s ,
a and
~ - L l a l a c t o s i d a s e s ,i n v e r t a s e o r s u c r a s e ,
p r o t e a s e s , a s p a r a g i n a s e , g l u t a m i n a s e , u r e a s e , a n d ~ h o s ~ h a t a s ea r) e
t h o s e a c t i v e l y s e c r e t e d t o t h e s o i l e n v i r o n m e n t by a wide r a n g e o f
m e t a b o l i c a l l y a c t i v e c e l l s (mostly b a c t e r i a , fungi and actinomycetes)
and l i v i n g p l a n t r o o t s ( S k u j i n s , 1967).
They a r e i n v o l v e d i n t h e
breakdown o f h i g h m o l e c u l a r w e i g h t compounds t h a t c a n n o t p e r m e a t e
the c e l l rmbrane.
Some o f t h e s e enzymes a c c u m u l a t e a t h i g h back-
ground l e v e l s i n t h e s o i l .
The enzyme l e v e l s i n s o i l are known t o b e
-11r e l a t i v e l y c o n s t a n t compared t o f l u c t u a t i n g s e a s o n a l o r l o c a l c h a n g e s
i n m i c r o b i a l numbers ( S k u j i n s , 1967; P a u l s o n a n d K u r t z ,
1969).
L i k e w i s e , h i g h l e v e l s o f t h e s e enzymes c a n o c c u r where m i c r o b i a l
p r o l i f e r a t i o n is suppressed ( H e r l i h y , 1972).
O t h e r e x t r a c e l l u l a r enzymes are t h o s e r e l e a s e d t o t h e s o i l
e n v i r o n m e n t by d e a t h a n d decay o f p l a n t a n d a n i m a l t i s s u e .
I t is
d i f f i c u l t t o d e c i d e w h e t h e r a n enzyme, e v e n o n e i n p u r e c u l t u r e ,
i s t r u l y e x t r a c e l l u l a r o r w h e t h e r i t h a s been r e l e a s e d upon a u t o l y s i s
of c e l l s .
S o i l enzymes have d i f f e r e n t ) o r i g i n s ( m i c r o o r g a n i s m s , p l a n t
r o o t s a n d s o i l a n i m a l s ) and e a c h enzyme h a s i t s own c h a r a c t e r i s t i c s .
Thus s o i l shows a c t i v i t y o v e r a b r o a d pH r a n g e .
This allows f o r a
u n i v e r s a l i t y o f s o i l enzymes; t h a t i s , t h e a b i l i t y o f s o i l t o have
c a t a l y t i c a c t i v i t y under c o n d i t i o n s impossible f o r b a c t e r i a l f l o r a ,
fungi. o r t h e r o o t system o f p l a n t s a c t i n g s e p a r a t e l y ( ~ u ~ r e v i c h
and Shcherbakova,
1971 1.
Lennox ( 1 9 5 3 ) , among o t h e r s h a s shown t h a t enzymes are r e l e a s e d
t o t h e s o i l medium i n a c e r t a i n s e q u e n c e :
f i r s t , t h e carbohydrases
a n d p h o s p h a t a s e s , t h e n t h e p r o t e a s e s a n d esterases, a n d f i n a l l y
catalase.
S i n c e many o r g a n i s m s c o n t r i b u t e t o t h e s o i l enzyme p o o l ,
i t i s d i f f i c u l t t o a s s o c i a t e a n enzyme m o l e c u l e i n t h e s o i l w i t h a
p a r t i c u l a r o r g a n i s m t h a t produced i t .
Also, t h e r e l a t i v e a c t i v i t i e s
o f enzymes o f d i f e r e n t o r i g i n a r e unknown
add,
1978).
It is
anticipated that in many cases the contributions made by
different organisms to the total activity of accumulated enzymes
in soils may vary, not only seasonally and with type and age
of plant cover but also with the treatment of soils between
sampling and assay,
Only a few enzymes have been extracted from soils due to
the resistance of soil enzymes to extracting agents.
Thus the
range of enzymes in soil commonly listed (about fifty), is not
derived from studies of isolated enzymes catalyzing clearly
defined reactions, but includes enzymes which reasonably but
b
not conclusively may have contributed to the activity measured
add, 1978). Soil enzymes most frequently studied are
oxidoreductases (e.g.
dehydrogenases, catalase, peroxidase,
glucose oxidase and other oxidases) and hydrolases (e.g.
carboxylesterase, lipase, phosphatase, sulphatase, urease,
amylase, cellulase and invertase).
Studies of catalase,
dehydrogenase, invertase, protease, urease and phosphatase
activities in soils account for most publications on soil enzymes
(~kujins, 1978).
1.2
State of Enzymes in the Soil
The precise physical state of the extracellular enzymes
in soil is not clearly understood.
Rurns et
- a1
- (1972) suggest
that enzymes are physically and chemically immobilized within
the discontinuous organic colloidal material which is itself
associated with soil clay particles (the organomineral complex):
an imnobilization that occurs during humic matter genesis when
exoenzymes and endoenzymes from active and lysed cells become
trapped.
It is apparent that abiontic enzymes are adsorbed on
surfaces of the colloidal soil particles and are in some way
covalently bound to inorganic and organic macromolecular components.
HcLaren (19751, therefore suggested that soil can be looked on as a
system of hunus
-
and
clay-immobilized enzymes.
Clay and silt fractions are more effective in adsorbing
extracellular enzymes than sand particles ( ~ o m i n eand Kobayashi,
1966).
Thus the underlying rock might affect soil enzyme activity.
The attachment of the enzymes to clay and silt particles is partly
by ion exchange and partly by nonionic bonding.
Many enzymes become
adsorbed between the clay lattices where both their accessibility to
microbial proteases and their catalytic activity are generally,
although not exclusively reduced.
These interlammelar proteins
virtually act as molecular calipers (Burns, 1978).
Extracellular
enzymes become bound to humic compounds either by covalent linkage
during formation of the humic polymers or perhaps by ionic or hydrogen
bonding to the preformed humic compounds in such a way that some
enzyme activity is retained and the enzymes acquire enhanced stability
to biological attack.
Some enzymes may also become bound to naturally
occurring compounds such as lignin, tannins and melanin pigments of
plant origin ( h d d and Butler, 1975).
The physical and chemical association of extracellular enzymes
- 14with s o i l c o l l o i d a l p a r t i c l e s renders t h e p r o t e i n molecules
more s t a b l e t o e l e v a t e d t e m p e r a t u r e s a n d pH f l u c t u a t i o n s t h a n
s i m i l a r homogeneous enzymes s t u d i e d
v i t r o , and t h e s e enzymes
are a l s o i n a c c e s s i b l e t o i n h i b i t o r y a n d e x t r a c t i n g a g e n t s ,
They are q u i t e r e s i s t a n t t o h y d r o l y s i s by m i c r o b i a l p r o t e a s e s
a n d t o d e n a t u r a t i o n i n a s o i l e n v i r o n m e n t (Ladd and B u t l e r ,
1969).
E x t r a c e l l u l a r enzymes t h e r e f o r e p e r s i s t i n s o i l s as
a c t i v e m o i e t i e s f o r long p e r i o d s a f t e r t h e o r i g i n a l s o u r c e
h a s b e e n removed o r d e s t r o y e d ( S k u j i n s a n d McLaren, 1968;
Ladd, 1978) a n d t h e i r a c t i v i t i e s are i n d e p e n d e n t o f t h e m i c r o b i a l
p o p u l a t i o n (Paulson and Kurtz, 1969).
Thus, measured enzyme
a c t i v i t i e s do n o t i n d i c a t e t h e e x i s t i n g m i c r o b i a l a c t i v i t y i n
t h e s o i l b u t r e f l e c t t h e accumulat.ed enzyme a c t i v i t y due t o
microor&misms t h a t had e x i s t e d i n t h e s o i l f o r a c o u p l e o f
years.
1.3
F a c t o r s A f f e c t i n g Enzyme A c f i v i t y W i t h i n t h e S o i l Environment
The a b i o n t i c enzymes i n s o i l e x i s t i n a h e t e r o g e n o u s
s y s t e m i n which t h e e n z y m a t i c r e a c t i o n s t a k e p l a c e a t t h e
solid/liquid interfaces.
The m i c r o e n v i r o n m e n t o f t h e s e extra-
c e l l u l a r enzymes i s r e s t r i c t e d on o r w i t h i n t h e s o i l c o l l o i d a l
p a r t i c l e s and it i s i n t h i s molecular environment t h a t t h e
c a t a l y s i s occurs.
Among t h e many f a c t o r s t h a t may a f f e c t enzyme
a c t i v i t i e s i n s o i l s , c r o p p i n g h i s t o r y , s o i l amendments,
- 15vegetation cover, agricultural chemicals, industrial pollutants,
soil fixation and ,some climatic factors have special influences
(Kiss &&., 1975; Tabatabai, 19821,
The effects v
v with specific enzymes and depend, among other
factors, on the soil type, dose of chemical, type and age of
d .,1975).
vegetation cover and conditions of study ( ~ i s s d
Soil
moisture is important in regulating soil enzyme activities since
biochemical reactions take place in solution,
Studies on the extra-
cellular enzyme activities in ecosystems indicate seasonal variations
of same enzyme levels in soils+arising from rainfall sequences
(~tojonavic,1959;Cooper, 1972: Neal Jr. 1973). However, the molecular
environment in which the immobilized extracellular enzymes act
(i.e.
soil particle surfaces) maintain a thin water film around it
that enables enzyme catalysis to take place even when the moisture
status of the bulk soil solution does not permit the survival of
plant roots and microorganisms.
Like other biochemical reactions
in soils, enzyme activities are associated with organic matter
distribution profile and generally decrease with depth,
activities are detectable between 0
-
Soil enzyme
15 cm layer of most soils
(Skujins, 1567).
Almost invariably the specific activity of an enzyme is lowered
as a result of the enzyme becoming attached to or entrapped within an
insoluble organic matrix and inorganic soil particles (Lkozdowicz,
1971; Ladd and Butler, 1975).
Amorphous allophane, halloysite and
montmorillonite are known to reduce the hydrolysis of starch by
- 16/-amylase
between 'j%
t o 3&
a n d Kobayashi, 1966).
o f t h e f r e e enzyme a c t i v i t y ( ~ o m i n e
S i l t and c l a y p a r t i c l e s f i x e x t r a c e l l u l a r
enzymes w h i l e t h e s a n d f r a c t i o n p l a y s o n l y a minor r o l e ( ~ a i ~ ,
1955).
Hence t h e t y p e o f p a r e n t m a t e r i a l c a n have a n e f f e c t on
s o i l enzyme a c t i v i t i e s .
The i n h i b i t o r y e f f e c t o f s o i l f i x a t i o n on
e x t r a c e l l u l a r enzyme a c t i v i t y a r i s e s from s t e r i c r e s t r i c t i o n s
imposed by t h e carrier ( s o i l p a r t i c l e s ) , e i t h e r a s a r e s u l t o f
r e s t r i c t e d movement o f s u b s t r a t e t o t h e o t h e r w i s e a v a i l a b l e a c t i v e
s i t e o f t h e enzyme o r b e c a u s e o f t h e o r i e n t a t i o n o f t h e enzyme i n
r e l a t i o n t o t h e carrier s u r f a s e .
S o i l p a r t i c l e s t h a t bind s o i l
enzymes impose s t e r i c r e s t r i c t i o n s which r e s u l t i n slow d i f f u s i o n
of o r g a n i c s u b s t r a t e s t o t h e immobilized enzyme s i t e (Ladd a n d
Butler,
1975), which i s t h e m o l e c u l a r environment of t h e enzyme.
Thus, t h e s u b s t r a t e c o n c e n t r a t i o n i n t h e immediate v i c i n i t y o f t h e
a t t a c h e d enzyme i s lower t h a n t h a t i n t h e b u l k s o i l s o l u t i o n .
S t u d i e ~show t h a t when s u b s t r a t e d i f f u s i o n becomes r a t e - l i m i t i n g ,
1.
a n i m n o b i l i z e d enzyme does n o t a t t a i n i t s maximum v e l o c i t y (V
max
Thus methods t o d e t e r m i n e t h e k i n e t i c c o n s t a n t s ( k m a n d ~ m a x b
) a s e d on t h e
Wichaelis-Menten e q u a t i o n u s e d i n homogenous e x p e r i m e n t a l s y s t e m s
do n o t a p p l y t o t h e s o i l h e t e r o g e n o u s system ( ~ a b a t a b a i , 1982).
The e x p e r i m e n t a l c o n d i t i o n s a n d t h e s o i l b e h a v i o u r i n t r o d u c e
parameters t h a t a r e not measurable and cannot be included f o r t h e
d e t e r m i n a t i o n o f krn v a l u e s and t h e maximum
Kurtz,
1970: T a b a t a b a i a n d Rremmer,
1971).
v e l o c i t i e s ( P a u l s o n and
In a homogenous system, for enzyme
on
a
substrate
in relatively
,
concentration [E]
acting
large concentrations in solution,
we have the familiar Michaelis-Menten equation (Lehninger, 1982):
for the disappearance of substrate of concentration
action of the enzyme.
[s]
under the
Km is the substrate concentration at which
this rate is one-half the maximum rate and is characteristic of the
soluble enzyme-substrate system.
*
In the soil heterogenous system
where the enzyme is in excess the corresponding equation becomes
(McLaren and Skujins, 1971):
The constant K
3
is characteristic of the decomposition of the E-S
complex and may be independent of the state of the substrate (e.g.
sol or gel).
However, km involves the rate of formation of the E-S
complex and this can depend markedly on the state of the substrate.
Binding to soil particles lowers the km of soil enzymes, hence
increases their affinity to substrates.
At
interfaces the surface pH can be important in governing the
rate of soil enzyme activities.
The surfaces of soil colloids
selectively adsorb cations which include the biologically important
+
H ions.
Thus the pH in the immediate vicinity of the irmobilized
- 18the
enzymes is lower than inpulk soil environment (the soil solution).
The observed pH maxima of the extracellular enzymic activities are
considerably higher than the corresponding homogenous enzymes.
For
example, the optimum pH of cellulase activity in a soil extract
was found to be 5-6 (Ha~ano,19861, while cellulase activity of
Aspergillus niaer is reported to be optimal st a b a u t pH '4.5
and Stone, 1965).
(Clarke
Any biocatalytic process in soil involves a
nlnnber of simultaneously participating catalysts with different
origins.
This is the reason that soil shows activity over a broad
pH range (Kuprevich and Shcherbakova, 1971).
Generally soils low in cation exchange capacity (CEC) and low
in organic matter display lower degree of enzymatic activity (Kiss
et
-
aJ,,
1978).
As a rule, highly cultivated soils possess a higher
level of enzymatic activity.
Overgrazing and erosion decrease
activity (Skujins, 1973) probably due to loss of organic matter,
Since plant root exudates contribute to the soil enzyme pool, the
type of vegetation cover will affect extracellular soil enzyme
activity (ROSS, 1965).
Enzymatic activities in diverse types of soils differ widely.
The differences in level of enzymatic activity are caused primarily
by the fact that every soil type, depending on its origin and
developmental conditions, is distinct from every other in its content
of organic matter, in the composition and activity of living organisms
inhabiting it, and, consequently, in the intensity of biological
processes thus, each type of soil has its own inherent level of
enzymatic activity ( ~ u ~ r e v i cand
h Shcherbakova, 1971).
Specific
characteristics of enzymes must be associated with different
origins.
The production of soil enzymes from different sources
allows for a universality of the extracellular enzymes, that
is, the ability of soil to have catalytic activity under
conditions impossible for bacterial flora, fungi, or the root
avstem of plants acting separately.
1.4
Soil Enzymes and ~iolo~ical/~ertility
Index of Soilsb
Biological activity of soil is usually most conveniently
determined by measuring
(i)
the quantity of carbon dioxide produced, i.e.,
its respiration;
(ii)
(iii)
the activities of certain extracellular enzymes; and
the total number of microorganisms
-
(microbial count).
For measuring biological activity of a soil,,Hoffmann (1963),
considered the enzymatic method more useful than determining the
number of microorganisms or measuring respiration.
A11 the
metabolic transformations in the soil effected by its active
population (billions of bacteria, fungi, algae, actinomycetes,
protozoa, etc) are mediated almost exclusively by enzymes.
Thus the most essential index of biological activity in soil
is its enzymatic activity (Skujins, 1978).
-20Various tests have been used in correlating extracellular
enzymatic activities with soil fertility and with microbial activities
iological indexttin soils
for the purpose of establishing a Itb
and consequently, a "fertility index" of soils usable for practical
purposes in agriculture (Skujins, 1978).
Such expectations were
expressed by a number of investigators in the 1950s.
Unfortunately,
the observations produced conflicting and confusing data and some
methodologies were questionable.
Hofmann and Seegerer (1950),
suggested that invertase activity would indicate the quality of
soil and would be usable as a measure of its fertility.
Lajudie
b
and Pachon (19%), suggested that soils may be classified according
to their proteolytic activity and that such classification did agree
with the biological activity and the apparent fertility.
Moureaux
(1957). examined invertase activity and populations of N -fixing
2
and nitrifying organisms in a wide range of soils.
The results
showed significant variations in activities according to soil type,
topography and plant cover.
The Moureaux values generally reflected
the producticity of a given soil and it was suggested that the use
of biological tests to compliment physical and chemical analysis of
soils was justified for fertility determinations.
Maliszewska (1969),
compared biological activities of different soils and suggested that
soil respiration, proteolytic activity and cellulolytic activity
were the most appropriate parameters for correlating soil fertility.
As
more information on abiontic enzyme activities in soils
became available, correlations with soil fertility became more difficult
t
-21t o support and gnenerali-tions
This is
more d i f f i c u l t t o make.
b e c a u s e enzyme a c t i v i t y i n s o i l s , a s d e t e r m i n e d i n v i t r o i s a
m a n i f e s t a t i o n o f s e v e r a l b i o l o g i c a l parameters i n s o i l including
m o u n t a n d t y p e o f o r g a n i c matter, number and t y p e o f m i c r o o r g a n i s m s ,
p l a n t root a c t i v i t y , s o i l pH, s o i l m o i s t u r e , etc.
Enzymatic a c t i v i t i e s have been examined i n s o i l s o f most
g e o g r a p h i c r e g i o n s o f t h e world a n d c e r t a i n s i m i l a r i t i e s have emerged.
However, t h e g e n e r a l d e c r e a s e i n e n z y m a t i c a c t i v i t i e s w i t h d e p t h o f
s o i l may be w e l l c o r r e l a t e d b u t a h o r i z o n t a l d i s t r i b u t i o n c o r r e l a t i o n
i s seldom e v i d e n t ( S k u j i n s ,
1978).
Each s o i l t y p e p r e s e n t s i t s own
b
e n z y m a t i c p a t t e r n a n d most o f t e n , t h e e n z y m a t i c a n d o t h e r b i o c h e m i c a l
and s o i l m i c r o b i o l o g i c a l a c t i v i t i e s c a n n o t be c o r r e l a t e d .
Typically,
e n z y m a t i c a c t i v i t i e s do n o t c o r r e l a t e w i t h s o i l r e s p i r a t i o n v a l u e s ,
n o r w i t h m i c r o b i a l numbers ( S k u j i n s , 1978; Pancholy a n d R i c e ,
although t h e r e a r e always exceptions.
1973b),
C u l t i v a t i o n most o f t e n i n c r e a s e s
v a r i o u s a c t i v i t i e s a n d a s a r u l e , s o does t h e a p p l i c a t i o n o f
f e r t i l i z e r s a n d o r g a n i c amendments.
Khan (19701, re-examined e n z y m a t i c
a c t i v i t i e s i n s o i l s as i n f l u e n c e d by v a r i o u s c r o p s a n d f e r t i l i z e r s
and n o t e d t h a t i n v e r t a s e , p h o s p h a t a s e and c a t a l a s e a c t i v i t i e s i n t h e
s u r f a c e h o r i z o n i n c r e a s e d when v i r g i n s o i l s w e r e c u l t i v a t e d .
To
c o r r e l a t e enzyme a c t i v i t i e s w i t h c r o p y i e l d s , Yaroshevich (19661,
examined s o i l s s u b j e c t e d t o f i f t y y e a r s '
application.
rotated f e r t i l i z e r
H e showed t h a t c o n t i n u e d u s e of manure i n c r e a s e d s o i l
r e s p i r a t i o n a n d e n z y m a t i c a c t i v i t y , whereas i n o r g a n i c f e r t i l i z e r s
o f e q u i v a l e n t n u t r i e n t v a l u e had t h e o p p o s i t e e f f e c t .
I n a number
-22-
test plots where soils had the same fertility value for plants,
enzyme activities varied according to the fertilizer regime and had
no obvious correlation with the yield of sugar beet, the test plant.
In a short-term fertilizer trial, Galstyan (1963), concluded that
in most cases higher enzyme activity was associated with higher
crop yield and vice versa,
However, in a long-term fertilizer trial
on a brown soil, soil amylase activity measured in the final year,
showed no correlation with the crop (maize) yield achieved in that
year.
For example, the highest activity was registered in the
unfertilized soil with very low crop yield.
Soils fertilized with
N P K had the highest crop yields but only moderate enzymatic activity,
Till now it has not been possible to obtain a fertility index
by the use of abiontic soil enzyme activity values.
It is evident
that enzymes are substrate specific and individual enzyme measurements
cannot reflect the total nutrient status of the soil (Skujins, 19781,
Individual soil enzyme measurements, however, might answer questions
regarding specific decomposition processes in the soil or questions
about specific nutrient cycles,
For example, it is of value to know
the proteolytic activity characteristics in soil if one is concerned
with the nitrogen cycle, or to examine phosphatase and arylsulphatase
activities and to correlate them with phosphate and sulphate availability respectively from soil organic sources.
Similarly, urease
is important agriculturally as an enzyme which might limit the
nitrogen available to plants from fertilizers or natural sources,
Amylase, cellulase and other carbohydrases might indicate decomposition
rates of litter and hence rate of mineralization of organic matter.
It should not be concluded, however, that a "fertility index"
for soils based on soil biochemical properties, might not eventually
become available, the necessary parameters and the correlative
relationships are just not yet apparent (Skujins, 1978).
It is
interesting to note that soils or other porous rocks that have lost
or have not acquired an enzymatic complex are unsatisfactory for
successful development of higher plants; their fertility is negligble.
Although there is no strict covelation between abiontic soil enzyme
activities and degrees of soil fertility, weakly active soil fail
to provide higher plants with qualities characteristic of crops
grown on rich, biologically highly active soil. (Skujins, 1967).
It is hoped that soil enzyme studies will eventually indicate
the degree of mineralization of organic matter.
This will enable
us to allow for plant nutrients that will be released over a growing
season when making fertilizer recommendations.
In most soil-test
laboratories, no test for available nitrogen is being used at the
present time.
This is because most of the nitrogen is stored in the
organic fraction, and there is no convenient, rapid test to measure
the amount that will be mineralised over the growing season (standfort
Also soil enzyme activity has been considered of possible value
as an index of biochemical activity in soil classification.
(1%6),
Ross
suggested that determinations of enzyme activities in the
-24t o p s o i l c a n be o f some v a l u e i n t h e d e s c r i p t i o n o f s o i l s , p a r t i c u l a r l y
o f d i f f e r e n t major groups of zonal s o i l s .
A t p r e s e n t i t is n o t
p o s s i b l e t o c l a s s i f y s o i l s according t o t h e degree o f enzymatic
a c i t i v i t i e s p r e s e n t b e c a u s e o f t h e enormous m u l t i t u d e o f s o i l s a n d
t h e f a c t t h a t o n l y i s o l a t e d c a s e s h a v e been s t u d i e d .
2. S o i l Polysaccharidases (Carbohydrases)
S o i l p o l y s a c c h a r i d a s e s o r c a r b o h y d r a s e s a r e enzymes w h i c h
c a t a l y s e t h e hydrolytic depolymerization of polysaccharides i n t h e
soil.
The most a b u n d a n t o r g J n i c compounds i n n a t u r e are p o l y s a c c h a r i d e s .
A l a r g e p a r t o f t h e p l a n t r e s i d u e s and a s m a l l e r p a r t o f t h e animal
r e s i d u e s t h a t f i n d t h e i r way i n t o s o i l a r e p o l y s a c c h a r i d i c .
I
Con-
I
- s e q u e n t l y , t h e d e c o m p o s i t i o n o f c a r b o h y d r a t e p o l y m e r s a n d t h e subsequent m i n e r a l i z a t i o n o f t h e p r o d u c t s have a s p e c i a l s i g n i f i c a n c e
i n the biological
on o u r p l a n e t .
c y c l i n g of carbon and t h u s t h e p e r p e t u a t i o n o f l i f e
I t is t h e r e f o r e l o g i c a l t o a t t r i b u t e a similar
significance t o t h e s o i l polysaccharidases ( K i s s e t al.,
1975)-
P o l y s a c c h a r i d e s a r e t h e main e n e r g y a n d c a r b o n s o u r c e s f o r s o i l
microorganisms,
b u t t h e y are t o o l a r g e t o permeate t h e m i c r o b i a l c e l l
membranes ( A l e x a n d e r , 1 9 7 7 ) -
Thus t h e s e o r g a n i s m s s e c r e t e enzymes
i n t o t h e s o i l environment t o degrade t h e c a r b o h y d r a t e polymers.
P o l y s a c c h a r i d a s e s a r e t h e r e f o r e t r u e e x t r a c e l l u l a r enzymes ( K i s s
1978: G l e n n ,
1976).
et
Polysaccharidase a c t i v i t y i n s o i l , l i k e t h a t of
o t h e r enzymes, r e s u l t s from a c c u m u l a t e d p o l y s a c c h a r i d a s e s a s w e l l a s
a.,
-25the activity of proliferating microorganisms.
By definition, accu-
mulated polysaccharidases are regarded a- cnzymcs present and active
in a soil independent of immediate microbial proliferation ( ~ i s s
et d.,1978).
-
Sources of this %ackgroundl enzyme level are primarily
microbial cells (Hofmann, 1963; Alexander, 1977) although some, no
doubt, originate from plant roots (Hayano, 1986) and animal residues
(Okada et
d., 1966).
Polysaccharidases have been of interest to soil enzymologists
because they degrade polysaccharides which are the most abundant
organic canpounds in nature.
ThCy have been a favourite hunting
ground for soil enzymologists since the enzymatic reaction products
(most often glucose) are easily discernible in reaction mixtures where
the substrate usually is insoluble and neither the products nor
substrates react with soil particles (Hofmann and Seegerer, 1950).
The accumulated polysaccharidases so far found in soil include: o(and @-amylases, cellulase, lichenase, inulase, laminarinase, xylanase,
extrinase, dextranase, levanase, invertase, polygalacturonase
(pectinasel, 4-galactosidase, and
gentiobiase
/-glucosidase
e
.c .
emulsi n, c e l l ~ b i a s e
(Kiss et al., 1975; Skujins, 1976).
Under natural soil conditions the polysaccharidases, like other
enzymes, are continuously being synthesized and accumulated, inactivated
and decomposed.
Fluctuations in measured enzyme activity reflect the
momentary ratio between these opposite processes.
The factors influencing
these changes include climatic conditions, soil properties, vegetation
-%-
cover and agricultural and silvicultural techniques ( ~ i &
s ~
d.,1978).
All these factors are interrelated and their
effects vary with individual soil enzymes.
Ross (1965) studied
carbohydrases (invertase and amylase) and verified that enzymatic
activities were considerably influenced by vegetation cover,
period of sampling and depth of soil, while the clay content
had less effect.
Variations in annual rainfall and soil organic
carbon when considered together but not separately, explained
much of the variability in sucrose hydrolysing activities, which
seem to be related more to poi1 composition than to its organic
matter content.
Similar variations in activities of other soil
enzymes have been shorn, and the lack of correlation among the
enzymes and specific soil properties has become more evident.
2.1
Influence of Crops on the Polysaccharidase Content of Soils
Crop plants, like other higher plants, contribute to the
content of polysaccharidases and other enzymes in soil both
directly and indirectly (ROSS, 1965; Hayano, 1986).
Their direct
contribution is by way of plant polysaccharidases (secreted in
root exudates), whilst their indirect contribution is connected
with the microbial decomposition of plant residues during which
microbial polysaccharidases are synthesized.
Among plant organs
the roots are the most important sources of the soil enzymes.
In addition to the endoenzymes contained in root residues,
extracellular enzymes secreted by living roots may make a
significant contribution t o the t o t a l activity.
The i n f l u e n c e o f a g i v e n p l a n t o n t h e p o l y s a c c h a r i d a s e
c o n t e n t o f s o i l c a n b e a s s e s s e d by c o m p a r i n g enzyme a c t i v i t y
i n t h e r h i z o s p h e r e and t h e non-rhizosphere
s o i l ; t h i s influence
may very a c c o r d i n g t o t h e a g e o f t h e v e g e t a t i o n a n d t h e s e a s o n
(Ross,
1%5; P a n c h o l y a n d R i c e , 1 9 7 3 a ) .
Different crop species
may i n f l u e n c e t h e s o i l p o l y s a c c h a r i d a s e c o n t e n t t o a d i f f e r e n t
t
extent (Kiss e
&.,
1978; Hayano, 1 9 8 6 ) .
It is a l s o possible
t h a t t h e n a t u r e o f t h e p r e v i o u s c r o p affects t h e c u r r e n t
polysaccharidase content of s o i l .
e
2.2
Significance of Polysaccharidases i n t h e SoilThe b i o l o g i c a l s i g n i f i c a n c e o f p o l y s a c c h a r i d a s e s i n t h e
s o i l may be s t a t e d as f o l l o w s ( K i s s e t a l . ,
1.
1975):
They decompose p l a n t l i t t e r a n d i n c r e a s e t h e r a t e o f
humus f o r m a t i o n a n d m i n e r a l i z a t i o n o f s o i l o r g a n i c
matter.
Thus t h e y p l a y a p o s s i b l e r o l e i n i m p r o v i n g
soil fertility.
2.
They h e l p i n t h e c y c l i n g o f c a r b o n i n t h e s o i l .
Soil
p o l y s a c c h a r i d a s e s are e s s e n t i a l i n t h e e c o l o g i c a l
b a l a n c e between o r g a n i c r e s i d u e s a n d a t m o s p h e r i c c a r b o n
d i o x i d e a n d t h u s t h e p e r p e t u a t i o n o f l i f e On o u r
planet.
3.
They i n c r e a s e
.
biological l i f e i n the soil.
P o l y s a c c h a r i d e s are t h e c h i e f s o u r c e s of carbon and
-28energy to soil microorganisms (various fungi, bacteria
and a~tinom~cetes). After hydrolysis by polysaccharidases,
the sugars released are used by soil microflora, whose
activities
fertility.
-
particularly nitrogen fixation, increase soil
The monosaccharides are also of potential
importance in the nutrition of plants, egg. xyloketose is
known to help in seed germination and in the elongation
of roots.
4.
Polysaccharidases hydrolyse some complex polysaccharides
to
shorter
ones capable of binding inorganic soil
b
particles (through hydrogen bonding and Van der Waals
attraction) into stable aggregates in the fonnation of a
desirable soil structure.
5.
Polysaccharidases are potential tools in extraterrestial
life exploration.
The presence of polysaccharidases in
other planets would be an indication of the presence of
organic compounds there.
2.3
Characteristics of Soils Based on Their Levels of Polysaccharidases
Levels of polysaccharidases might indicate decomposition
index of soils, i.e.,
the turnover rate of plant
litter in soils ( ~ i s set al., 1978).
Khan (19701, correlated the activity of polysaccharidases
with the organic matter content of soils.
(c)
L e v e l s o f p o l y s a c c h a r i d a s e s h a v e b e e n shown t o b e r e l a t e d
t o m i c r o b i a l numbers a n d m e t a b o l i c a c t i v i t y o f s o i l s .
Sometimes however, peak enzyme a c t i v i t i e s do n o t o c c u r
a t p e r i o d s o f peak m i c r o b i a l numbers ( ~ a n c h o l ya n d R i c e ,
197 3b)
.
Although t h e y may p l a y a v e r y i m p o r t a n t r o l e i n i m p r o v i n g
s o i l f e r t i l i t y ( K i s s et
&. ,
19751, t h e l e v e l s o f p o l y -
s a c c h a r i d a s e s are t h o u g h t n o t t o b e c o n c l u s i v e p a r a m e t e r s f o r
f e r t i l i t y index o f s o i l s .
F e r t i l i t y i n d e x f o r s o i l s based on
b i o c h e m i c a l p r o p e r t i e s a w p i t s f u r t h e r r e s e a r c h i n s o i l enzymology.
S o i l enzyme a c t i v i t i e s may however b e u s e d as s u p p o r t i n g
evidence t o o t h e r f e r t i l i t y index parameters.
Amylase a n d c e l l u l a s e a r e o f s p e c i a l s i g n i f i c a n c e i n t h e
c a r b o n c y c l e o f s o i l s ( A l e x a n d e r , 1977).
h e n c e are t e r m e d g l y c o s i d e h y d r o l a s e s .
They a c t on g l y c o s i d e s
Amylase a n d c e l l u l a s e
are s t r i c t l y e x t r a c e l l u l a r o r a b i o n t i c s o i l enzymes s i n c e t h e y
degrade t h e c a r b o h y d r a t e polymers, s t a r c h and c e l l u l o s e ,
r e s p e c t i v e l y , which c a n n o t p e r m e a t e m i c r o b i a l c e l l membranes.
2.4
S o i l Amylase (E.C.
3.2.1)
Amylase i s an enzyme t h a t c a t a l y z e s t h e h y d r o l y s i s o f
1 , 4 - g l u c o s i d i c bonds i n s t a r c h ( ~ e h n i n g e r , 1982).
Starch is
s e c o n d o n l y t o c e l l u l o s e a s t h e most common h e x o s e polymer i n
t h e p l a n t r e a l m ( ~ l e x a n d e r , 19771, hence i t s e r v e s a s a r e a d y
s o u r c e o f carbon and energy t o s o i l organisms.
Starch serves
- 30the plant as a storage product, and as such it is the major reserve
carbohydrate.
It occurs in large amounts in leaves carrying out
photosynthesis, but the polysaccharide is distributed in the xylem,
phloem, cortex, and pith of the stems of many plant species, as well
as in tubers, bulbs, corms, underground
(~lexander,1977).
rhizomes, fruits, and seeds
The starch component of these tissues disappears
rapidly when subjected to the activity of the soil community which
secrete starch-hydrolyzing enzymes (amylases) into the soil environment.
Since the starch molecule is too large to permeate microbial cell
membranes, amylase is a true extracellular enzyme, that is, it is
b
actively secreted into the soil environment ( ~ i s se t g . , 1978) where
it becomes bound to inorganic and organic soil particles (~kujins,
1976).
The abiontic amylase molecules accumulate in soil and can
persist within the soil environment for long irrespective of fluctuations of the organisms that produced them
add,
1978).
Apart from
microorganisms (bacteria, fungi, actinomycetes) that contribute to
the accumulated soil amylase pool, plant roots (root exudates) and small
soil animals
(Kiss &
(particularly earthworms) also play a significant role
e.,1975).
Plant starches contain two components, amylose and amylopectin
(l3o~erand Vanner, 1965).
The former has an essentially linear
structure built up of several hundred or more glucose units linked
together by an0(-(1+4)-~lucosidic
bonding.
In amylopectin, the
individual glucose units are likewise bound together byg-(1-4 4 )
linkages, but the molecule is branched and has side chains attached
-31through o(-(l-+6)-glucosidic
linkages.
In the soil environment
starch decomposition proceeds at a greater rate than the microbiologically induced losses of cellulose, hemicelluloses, and a variety
of' other polysaccharides (Alexander, 1977).
Under conditions of
limiting oxygen, a fermentation occurs with the formation of
appreciable lactic, acetic, and butyric acids.
The process of de-
gradation goes on at a good pace even under total anerobiosis, and
considerable methane may be evolved (Alexander, 1977).
Amylase is not a single enzyme, but an enzyme-complex consisting
of mainly three enzymes (Lehninger, 1982): 4-amylase (E.C.
b-amylase (E.C.
/-amylase
3.2.1.21,
and gfucoamylase (E.C.
3.2.1.1),
3.2.1.3).
(9(-1,~-glucanmaltohydrolase)is an exo enzyme that
hydrolyses d-1,4-glucan links in both amylose and amylopectin,
cleaving every second glucose-glucose bond from the end of the molecule
so as to remove successive maltose units (by inversion) from the
non-reducing ends of the chains. 1-amylase is capable of catalyzing
the hydrolysis of branch points of amylopectin, however, and a
residual dextrin fraction in addition to the disaccharide maltose
remains.
In contrast,&-amylase
(0(-1,4-~lucan 4-glucanohydrolase)
is an endo enzyme that acts randomly on the 1+
blinkage throughout
the amylose and amylopectin molecules to produce large amounts of
maltose and often small quantities of glucose and the trisaccharide
known as maltotriose (Lehninger, 1982).
Because it cannot attack the
branch points of amylopectin, high-molecular-weight dextrins containing
branches accmulate.
The prefix ofd-amylase denotes that the
- 32reducing group is set f r e e i n t h e
(
-1,4-glucan
-configuration.
Glucoamylase
h y d r o l a s e ) i s a l s o a n e x o enzyme which h y d r o l y z e s
s u c c e s s i v e g l u c o s e u n i t s from t h e n o n - r e d u c i n g e n d s o f t h e c h a i n s .
Some g l u c o a m y l a s e c a n c l e a v e b r a n c h p o i n t s o f a m y l o p e c t i n w h i l e
o t h e r s cannot s o t h a t d e x t r i n s accumulate.
The m a l t o s e , m a l t o t r i o s e , a n d low-molecular-weight
linear
o l i g o s a c c h a r i d e s g e n e r a t e d by t h e a m y l a s e s are c o n v e r t e d t o g l u c o s e
by m e d i a t i o n o f t h e enzyme
-glucosidase,
transformed u l t i m a t e l y t o glucose.
s o l u b l e a n d p e n e t r a t e t h e c e l l , *ere
s o t h a t t h e s t a r c h is
The s i m p l e s u g a r s are w a t e r t o be u s e d as e n e r g y s o u r c e s
f o r m i c r o b i a l g r o w t h and p r o t o p l a s m i c s y n t h e s i s ( ~ l e x a n d e r , 1 9 7 7 ) .
Amylase a c t i v i t y i n s o i l w a s f i r s t n o t e d by Hofman and S e e g e r e r
(1951).
A p p a r e n t l y , a l l t h r e e a m y l a s e s ( a - a m y l a s e , p-amylase a n d
g l u c o a m y l a s e ) e x i s t i n s o i l ( K u p r e v i c h a n d S h c h e r b a k o v a , 1971).
However, Hofmann a n d Hoffmann (39661, showed t h a t s o i l s c o n t a i n much
more p-amylase t h a n u - a m y l a s e .
According t o A l e x a n d e r (19771, a c t i v e
a - a m y l a s e i s p r o d u c e d by f u n g i ( n o t a b l y A s p e r g i l l u s , Fomes, F u s a r i u m ,
P o l y p a r u s a n d ~ h i z o p u s )a n d b a c t e r i a ( --B a c i l l u s , C l o s t r i d i u m ,
Chromohacterium, Pseudomonas, M i c r o c o c c u s , F l a v o b a c t e r i *
and Cytophaqa).
Among a c t i n c n n y c e t e s , N a c a r d i a , Micromonospora a n d S t r e p t o m y c e s a l s o
produce amylases.
p-amylases
are n o t common i n m i c r o o r g a n i s m s b u t are
d i s t r i b u t e d p r i m a r i l y among t h e h i g h e r p l a n t s a l o n g w i t h ()!-amylase
( ~ l e x a n d e r , 1977).
Glucoamylase i s p r o d u c e d m a i n l y by f u n g i .
Optimum a c t i v i t y o f a m y l a s e s o f v a r i o u s o r i g i n s o c c u r a t pH v a l u e s
from 4.0 t o 7.0 ( ~ u ~ r e v i ca nhd Shcherbakova,
1971).
-33Buffered s o i l suspensions and s o i l e x t r a c t s hydrolyse s t a r c h
u n d e r c o n d i t i o n s l i m i t i n g m i c r o b i a l p r o l i f e r a t i o n ; t h a t is i n d i c a t i v e
o f accumulated a m y l a s e s (Drobnik, 1955).
S o i l amylase i s i n d u c i b l e
(Drobnik, 1955) a n d d e c r e a s e s w i t h sample d e p t h a n d d e c r e a s e i n
o r g a n i c matter c o n t e n t (ROSS, 1968).
However, t h e a b i l i t y o f
microorganisms t o form a m y l o l y t i c enzymes depends on t h e t y p e o f
s t a r c h ( ~ l e x a n d e r , 1977) a n d c o n s e q u e n t l y , t h e t y p e of p l a n t m a t e r i a l
entering the s o i l .
Robert,
A c t i v i t i e s are i n f l u e n c e d by s e a s o n (Ross a n d
1 9 7 0 ) , s o i l t y p e , a n d by t h e n a t u r e o f t h e v e g e t a t i v e c o v e r
(Ross, 1966; 1968; Pancholy and p i c e ,
1W3a; Cortez &
e.,1975).
The a v e r a g e amylase a c t i v i t y o f l i g h t f r a c t i o n s o f s e v e r a l s o i l s
was found o n a w e t b a s i s , t o be e i g h t e e n t i m e s t h a t o f t h e heavy
f r a c t i o n s ( R o s s , 1975).
Kobavashi,
Amylase c a n be a d s o r b e d by c l a y s (Aomine and
1966) b u t t h e decomposition o f s t a r c h by t h e s o i l community
i s n o t a l v a y s a p p r e c i a b l y a f f e c t e d by t h e p r e s e n c e o f c l a y s ( ~ l e x a n d e r ,
1977)
-
Measured a m y l o l y t i c a c t i v i t y is based on i n c r e a s i n g t h e
r e d u c i n g power o f a s o l u t i o n o f t o l u e n e - t r e a t e d
starch.
s o i l s incubated with
The measured s o i l amylase a c t i v i t y i s a f f e c t e d by t h e
t r e a t m e n t of s o i l s between s r n p l i n g and a s s a y .
Amylase a c t i v i t y
d e c r e a s e s a f t e r a i r - d r y i n g o f s o i l s (ROSS, 1965) and a f t e r s t o r i n g
s o i l s a t d i f f e r e n t t e m p e r a t u r e s ( R o s s , 1965; Pancholy and R i c e , 1972).
A l l t h e t h r e e a m y l a s e s (o(-amylase,/-amylase
and g l u c o a m y l a s e )
a r e r e q u i r e d f o r t h e c o m p l e t e h y d r o l y s i s o f s t a r c h , t h u s t h e amylase
- 34a c t i v i t y u s u a l l y measured i n s o i l s r e p r e s e n t s t h e t o t a l a m y l o l y t i c
a c t i v i t y , t h a t i s , t h e a c t i v i t y o f t h e amylase-enzyme-complex,
r a t h e r t h a n o f i t s components.
S o i l C e l l u l a s e (E.C.
3.2.1.4)
C e l l u l a s e ~ - g l u c a n - 4 - g l u c a n o h y d r o l a s e ) i s t h e enzyme t h a t
catalyzes t h e hydrolysis o f theA-1,4-glucan
(L,ehninger,
1982).
links i n cellulose
C e l l u l o s e i s t h e most a b u n d a n t o r g a n i c
matter o n t h e e a r t h ' s c r u s t a n d p r o v i d e s most o f t h e c a r b o n a n d
energy t o s o i l f l o r a and fauna ( ~ l e x a n d e r , 1977).
Cellulose is
a c a r b o h y d r a t e , a polymer o f a l o n g c h a i n o f g l u c o s e u n i t s
linked byfl-(l+lt)
g l u c o s i d i c bonds ( D a v i e s e
t
m o l e c u l e s o f g l u c o s e l i n k e d by 8 - ( l + l t )
&.,
I%/*).
Two
bonds make u p c e l l o b i o s e
which is t h e r e p e a t i n g u n i t of c e l l u l o s e .
The m i c r o b i a l c e l l
i s impermeable t o t h e c e l l u l o s e m u l e c u l e s o t h e o r g a n i s m must
e x c r e t e c e l l u l a s e e x t r a c e l l u l a r l y i n o r d e r t o make t h e c a r b o n
and energy s o u r c e a v a i l a b l e .
The a b i o n t i c c e l l u l a s e a c t s
hydrolytically, converting t h e insoluble cellulose t o soluble
s u g a r s t h a t p e n e t r a t e t h e c e l l membrane.
Once i n s i d e t h e c e l l ,
?
t h e s i m p l e s u g a r s are o x i d i z e d a n d p r o v i d e e n e r g y f o r b i o s y n t h e t i c
r e a c t i o n s ( ~ l e x a n d e r , 1977).
Fungi, b a c t e r i a , a l g a e , a c t i -
nomycetes, and p r o t o z o a i n t h e g u t s o f t e r m i t e s produce c e l l u l a s e
enzymes ( ~ l e x a n d e r , 1 9 7 7 ) which e n t e r t h e s o i l e n v i r o n m e n t a n d
become a d s o r b e d o n o r g a n i c a n d i n o r g a n i c s o i l c o l l o i d s
-35(Drozodowicz, 1971).
Plant root exudates and digestive juice of
edible snail and earthworms contain cellulase enzymes (Kiss et
&., 1978; h d d , 1978).
These sources represent the sources of
cellulase enzyme that accumulate extracellularly in soil (~kujins
and Mchren, 1368; Skujins, 1976).
Although it has been assumed
for a long time a priori that cellulase exists in soil extracellularly,
the first attempt to show its extracellular existence in soil was
reported by Warkus (19551, who used cellophane pieces in toluenetreated soi1.
Cellulase production is the common denominator for cellulolytic
b
organisms.
The cellulosic material occurring in nature, however
varies greatly.
Those found in soil include non-living trunks,
stunps, felled timber, forest litter, non-woody plants, soft tissues
of fruits, roots and decaying grasses, and transformed debris of
all these organic materials mixed with soil material and offering
conditions for growth of selective microorganisms (Norkrans, 1967).
Most cellulolytic organisms are found among bacteria and fungi.
In
anaerobic soil environments bacteria are the outstanding cellulose
decomposers.
However, under aerobic soil environments bacteria
play an insignificant role in cellulolytic attack on wood; fungi
are more active (~lexander
, 1977).
Cellulase is not the name of a single enzyme, but an enzymecomplex consisting of three extracellular enzymes (C
1
component,
Cx component andp-glucosidase), all of which are required for the
complete hydrolysis of cellulose in the soil (~rozdowicz,1971).
-36The Cl component c a l l e d h y d r o c e l l u l a s e i s a p o o r l y c h a r a c t e r i z e d
enzyme t h a t a c t s on undegraded ( o r n a t i v e ) c e l l u l o s e t o form a
m o d i f i e d (more r e a c t i v e ) polymer,
I t i s assumed ( ~ o r k r a n s , 1967)
that t h i s a c t i v a t o r c e l l u l a s e component ( C 1) a c t s i n a way t o p e r m i t
a n i n c r e a s e d m o i s t u r e u p t a k e , h y d r a t i n g t h e n a t i v e c e l l u l o s e and
p u s h i n g apart t h e c l o s e l y packed c h a i n s , t o make t h e l i n k a g e s
a c c e s s i b l e f o r t h e a c t i o n o f t h e h y d r o l y t i c Cx component.
The Cx c m p o n e n t ( g - ( 1 - + 4 )
glucanase) does n o t hydrolyse
n a t i v e c e l l u l o s e b u t i n s t e a d c l e a v e s t h e p a r t i a l l y degraded polymers,
I t i s w i d e s p r e a d among f u n g i , b a c ) e r i a ,
1977).
and a c t i n o m y c e t e s ( ~ l e x a n d e r ,
T h e r e are two t y p e s o f g l u c a n a s e s ( ~ o r k r a n s , 1 9 6 7 ) :
(a
The endo+-(l-+lt)
glucanases hydrolyse t h e modified
c e l l u l o s e i n t e r n a l l y i n a more o r less random f a s h i o n .
They produce c e l l o b i o s e , v a r i o u s s h o r t o l i g o m e r s ,
and s o m e t i m e s g l u c o s e ,
(b)
The e x o $ - ( 1 1 4 )
g l u c a n a s e s remove s u c c e s s i v e g l u c o s e
u n i t s f r a n c e l l o b i o s e a n d s h o r t o l i g o m e r s from t h e
t e r m i n a l ends.
The last p h a s e i n t r a n s f o r m i n g c e l l u l o s e t o g l u c o s e i s
c a t a l y s e d by a t h i r d enzyme c a l l e d , 9 - ( 1 + 4 )
hy&olyzes
glucosidase t h a t
c e l l o b i o s e , c e l l o t r i o s e , and o t h e r low m o l e c u l a r weight
o l i g a a e r s t o glucose,
C e l l o b i a s e i s a n o l d name f o r b - g l u c o s i d a s e ,
but t h e term i s i n a p p r o p r i a t e because c e l l o b i o s e is n o t t h e o n l y
s u b s t r a t e ( ~ l e x a n d e r , 1977).
-37The cellulase caaplex found in soils is inducible in most
microorganisms and is synthesized in the presence of cellulose or
carbohydrates that are structurally similar to the polysaccharide,
cellobiose and a few other single carbohydrates containing glucose
in the molecule (Alexander, 1977).
In studies (Drozdowicz, 1971)
of the effect of enzyme substrate on cellulase activity, cellulose
(with or without (NH ) SO or K HPO ) was added to three organic
4 2 ft
2
It
soils; this induced an increase in cellulase contents during
incubation.
This increase fluctuated according to the soil type
and the N or P enrichment.
Ce~lulolyticactivity was limited by P
and N deficiency in the acid peat and by P deficiency in the calcic
peat.
In the mor of the podzol, neither
N
nor P deficiencies were
entirely responsible for the ccllulolysis (Kong and Dommergucs,
1972).
Cellulose and its products can be adsorbed by clay minerals.
But possibly of greater importance is the partial inactivation of
cellulase by certain clays (~oamineand Kobayashi, 19661, an effect
which has great significance because the enzyme system is extracellular, and therefore it can be altered in its activity by clays.
The enzyme inactivation and the substrate adsorption phenomenon may
account, at least in part, for the protective action of montmorillonite
clay on cellulosic materials subjected to microbial decomposition
(~lexander,1977).
Cellulase is also inhibited by melanin, a
constituent of the walls of a number of microorganisms (~lexander,1977).
-38The a s s o c i a t i o n o f c e l l u l o s e w i t h l i g n i n p a r t i c u l a r l y i n m a t u r e
p l a n t t i s s u e s d e c r e a s e s c e l l u l o l y t i c a c t i v i t y ( ~ o r k r a n s , 1967;
Uardrop, 1971)
.
A c t i v i t y o f t h e c e l l u l a s e system o r o f i n d i v i d u a l enzymes
i n t h e complex c a n be measured d i r e c t l y i n s o i l .
For t h i s
p u r p o s e , undegraded o r p a r t i a l l y degraded c e l l u l o s e , c e l l o p h a n e
o r c a r b o x y m e t h y l c e l l u l o s e (cMc) i s i n c u b a t e d w i t h s o i l f o r a
g i v e n p e r i o d and t h e q u a n t i t y o f s u g a r g e n e r a t e d
on^
and
Donmergues, 1972) o r t h e r e d u c i n g s u g a r c o n t e n t o f t h e f i l t r a t e
( P a n c h o l y a n d R i c e , 1973aJ d e t e r m i n e d .
The a c t i v i t y o f t h e enzyme
i s a f f e c t e d by t h e p r e s e n c e o f r o o t s a n d t h e t y p e o f p l a n t i n
t h e v i c i n i t y ( ~ l e x a n d e r , 1977).
a c t i v i t y r a n g e s from 3.9
(Roberge, 1978).
-
The optimum pH of s o i l c e l l u l a s e
7.5 b u t v a r i e s w i t h s o u r c e o f enzyme
No p u r e enzymes are i s o l a t e d from s o i l s hence
c r u d e s o i l e x t r a c t s ( s o i l s u s p e n s i o n s ) are o f t e n u s e d i n a s s a y s
j u s t as i n t h e d e t e r m i n a t i o n o f amylase a c t i v i t y .
C e l l u l a s e s h a v e been u s e d i n t h e food i n d u s t r y a s c e l l w a l l
d i s i n t e g r a t o r s i n o r d e r t o i n c r e a s e t h e d i g e s t i b i l i t y of
vegetable foods, e x t r a c t i b i l i t y of p r o t e i n s , f r u i t j u i c e s ,
e s s e n t i a l o i l s , etc.,
a n d i n t h e p h a r m a c e u t i c a l i n d u s t r y as a
d i g e s t i v e a i d ( N o r k r a n s , 1967).
I t i s hoped t h a t a c c u m u l a t e d
c e l l u l a s e s i n s o i l s w i l l h e a p o t e n t i a l s o u r c e of enzymes u s e d
i n t h e industry.
2,
Factors Influencing Amylase and Cellulase Activiti~
in Soils
Although soil microorganisms are thought to contribute
to most of the amylase and cellulase content of soils
(Hofmann, 1963), the total number of actinomycetes, bacteria,
and fungi are not always correlated with the activities of
these carbohydrases in soils (Pancholy and Rice, 1973b).
This
is because the accumulated amylase and cellulase activities
in soils are not largely due to the current microbial numbers,
but to the microbial activity that had existed in the particular
b
soil for the past years.
Apart from the type and number of
soil microorganisms, soil amylase and cellulase activities are
largely influenced by vegetation cover and agricultural
activities,
Reports of the various factors that influence the activity
gradients of amylase and cellulase in soils now follow.
2-6-1Rhizosphere Effect
Higher amylase and cellulase activities have been reported
in the rhiwsphere of several plants (winter rye, clover,
carrot, oat, vegetables, tomatoes, potatoes, sugar beets, maize
and other cereals, etc,) than in the non-rhizosphere soil
(Kiss et
d.,1978).
The activities increased with increasing
temperature (lo°C to 35OC) but decreased at lo°C or 35OC when
the humidity of the soil was increased from 60 to 90% of its
-40water h o l d i n g c a p a c i t y .
2.6.2
Type and Age o f V e g e t a t i o n Cover
R o s s (19651, found t h a t amylase a c t i v i t i e s w e r e h i g h e r
under legunes ( r e d c l o v e r and w h i t e c l o v e r ) t h a n under g r a s s e s
a t t h e same s i t e .
Depending on t h e c r o p , c e l l u l a s e a c t i v i t y
o f a sod-podzolic s o i l decreased i n t h e following o r d e r :
Amylase a n d c e l l u l a s e a c t i v i t y g r a d i e n t s w i t h a g e o f
v e g e t a t i o n depend on t h e t y p e o f p l a n t c o v e r .
Maximum v a l u e s
b
o f t h e enzymes o c c u r r e d i n t h e r h i z o s p h e r e o f young o a t p l a n t s
w h i l e minimum v a l u e s were found i n t h e f l o w e r i n g phase.
In
s p r i n g w h e a t , t h e a c t i v i t y i n c r e a s e d d u r i n g t h e development
o f t h e crop.
The i n c r e a s e was a t t r i b u t e d t o t h e growing r o o t
system s e r v i n g as s o u r c e o f amylase ( ~ i s se t a l . ,
2.6.3
2.6.3.1
1978).
A g r i c u l t u r a l Techniques
Tillage
s t aJ.,
Several r e p o r t s ( ~ i s %
1978) i n d i c a t e t h a t p l o u g h i n g
i n c r e a s e s t h e a c t i v i t y o f amylase a n d c e l l u l a s e enzymes.
C o n s e q u e n t l y , f o r improving b i o l o g i c a l a c t i v i t y i n lower
h o r i z o n s , p e r i o d i c m u l t i - s t a g e p l o u g h i n g i s recommended.
2.6.3.2
Drainage
D r a i n a g e a p p e a r s t o have o p p o s i t e e f f e c t s on amylase a n d
cellulase a c t i v i t i e s i n the soil.
Ross (19661, showed t h a t
a m y l a s e a c t i v i t y was s i g n i f i c a n t l y lower i n t h e d r a i n e d t h a n
i n t h e undrained s o i l .
S t u d i e s r e p o r t e d by K i s s e
t a l . (1978)
i n d i c a t e t h a t c e l l u l a s e a c t i v i t y increased with drainage.
The rate o f b i o c h e m i c a l c o n v e r s i o n s i n a s o i l i s d e t e r m i n e d
l a r g e l y by i t s w e t n e s s .
However, e x c e s s i v e s o i l m o i s t u r e
d e c r e a s e s m i c r o b i a l a c t i v i t y a n d hence t h e i r enzyme-producing
abilities.
Irrigation
b
Amylase ( R o s s , 1966) a n d c e l l u l a s e ( ~ i s et
s al.,
b o t h i n c r e a s e d on i r r i g a t i o n w i t h f r e s h w a t e r .
1978)
The p r e s e n c e
o f s t a r c h i n w a s t e w a t e r c a u s e d a r a p i d i n c r e a s e i n amylase
a c t i v i t y b u t a d e c r e a s e i n c e l l u l a s e a c t i v i t y (Ambroz, 1972).
S t a r c h i n d u c e s amylase p r o d u c t i o n i n m i c r o o r g a n i s m s b u t
b e c a u s e o f s i m i l a r i n t r a m o l e c u l a r bond t y p e s w i t h c e l l u l o s e ,
s t a r c h c o u l d a c t as a c o m p e t i t i v e s u b s t r a t e , hence c o u l d
decrease c e l l u l a s e activity.
2.6.3.4
F e r t i l i z a t i o n a n d Liming:
Amylase a c t i v i t y is h i g h e r i n s o i l s amended w i t h s t a r c h ,
sucrose, glucose o r with mixtures of these carbohydrates,
t h a n i n unamended s o i l s (Drobnik, 1955).
The h i g h e s t i n c r e a s e
o c c u r r e d i n samples t r e a t e d w i t h s t a r c h , t h e s u b s t r a t e f o r
amylase.
These o b s e r v a t i o n s w e r e i n t e r p r e t e d a s evidence o f
-142-
t h e i n d u c t i o n o f a m y l a s e s y n t h e s i s by t h e s o i l m i c r o o r g a n i s m s u n d e r
the influence of starch.
Thus Ross ( 1 9 6 6 ) ~ a n d P a n c h o l y a n d R i c e
(1973a), s u g g e s t e d that t h e i n f l u e n c e o f v e g e t a t i o n on s o i l a m y l a s e
a c t i v i t y is e x e r t e d through t h e s t a r c h c o n t e n t i n p l a n t r e s i d u e s
which, i n t u r n , induces m i c r o b i a l amylase s y n t h e s i s .
Other s t u d i e s
o f t h e e f f e c t o f o r g a n i c f e r t i l i z e r s on a m y l a s e a c t i v i t y r e p o r t e d
by K i s s ~ t a J . (19781, show t h a t a m y l a s e a c t i v i t y i n manured p l o t s
( g r e e n manure) w e r e h i g h e r t h a n i n t h e m a n u r e d o n e s , b u t n e a r l y
i d e n t i c a l i n t h e f a r m y a r d manure a n d s t u b b l e c r o p t r e a t m e n t s .
I n i n v e s t i g a t i o n s i n which s o i l enzyme a c t i v i t i e s were
c o n n e c t e d w i t h t h e h y g i e n i c p r o b l e m s o f i n c o r p o r a t i n g sewage s l u d g e
i n t o s o i l , Ukhtaaokaya ( 1 9 5 2 ) d e t e r m i n e d s o i l enzyme a c t i v i t y i n
e x p e r i m e n t a l p l o t s u s e d f o r f i n a l d i s p o s a l o f sewage s l u d g e .
Enzyme
a c t i v i t y w a s p e r i o d i c a l l y analysed, t o g e t h e r with o t h e r paramaeters
o f s a n i t a r y importance.
4
The r e s u l t s i n d i c a t e d t h a t s o i l a m y l a s e
a c t i v i t y g r e a t l y i n c r e a s e d immediately a f t e r t h e i n c o r p o r a t i o n of
Later, t h e a c t i v i t y d e c r e a s e d a n d w i t h i n o n e t o o n e a n d a
sludge-
half y e a r s r e t u r n e d t o t h e l e v e l i t had shown b e f o r e s l u d g e i n c o r -
poration.
The o n l y e x c e p t i o n w a s t h e s o i l t r e a t e d w i t h t h e h i g h e s t
amount o f s l u d g e .
Amylase a c t i v i t y may t h u s b e r e g a r d e d as a n i n d e x
of t h e self-purification
c a p a c i t y o f s o i l ( K i s s e t a l - , 1978).
R e s u l t s o f t h e e f f e c t s o f m i n e r a l f e r t i l i z e r s on a m y l a s e
a c t i v i t y have been c o n f l i c t i n g .
a.,( 1 9 7 8 )
t
However, K i s s e
t h a t a d d i t i o n of NPK ( N a p p l i e d as NaNO
3
o r NH NO
4
3
reported
while P and K
-43as K HPO ) on a short term basis ( 1 4 0 days) to acid soils (PH 4.2)
4
2
led to rapid increase in amylase activity.
However, continuous use
of NPK (60:60:60 kg/ha) in various combinations without liming had
a negative effect on the soil enzyme content,
Thus, under the
influence of the physiologically acid fertilizer (NH NO 1, soil amylase
4'
3
activity declined,
Liming of the continuously NH NO -treated acid
4 3
soil only partially restored amylase activity,
Overliming and the
subsequent increase in pH lowered the enzyme activity.
NH NO
3
Combinations of
plus K HPO with or without farmyard manure did not cause any
2
4
considerable changes in ,amylase activity as compared to the untreated
b
soil (Drobnik, 1957).
Dlagoveshchenskaya and hnchenko (197h), showed that cellulase
activity was constantly higher in soils fertilized with NPK than in
the unfertilized plots,
Soil cellulase activity was slightly higher
in the plots with NPK plus farmyard manure than in the NPK plots,
on both maize monoculture and crop rotation plots.
fertilization (90 kg/ha), as compared to the
145
High nitrogen
kg rate brought about
some decrease in cellulase activity.
et al., (1978) show that cellulose
Studies reported by Kiss induced cellulase production by microorganisms in soil, but carboxymethyl cellulose and cellobiose had no such effect.
The induction
was more pronounced with 1% than with 0.s cellulose powder,
The
importance of the cellulose content of plant residues for soil
cellulase activity was also emphasized by Pancholy and Rice (1973a)-
2.6.3.5
Effect of Pesticide Application
Herbicides greatly decrease soil amylase activity (Kelly
and Rodriguez-Kabann, 1975).
Most fungicides or insecticides
either have little (po3itive or negative) or no effect on
amylase activity (Ualasubramanian and Patil, 1968).
Cellulase is resistant to most herbicides (Cervelli
et &.,
-
1978).
Long term atrazine application (Cole, 1976)
led to only a slight decrease in cellulase activity.
Biology of Plant Litter Decomposition and Organic
Matter Content of Humid )rropical SoilsAlthough microbial and faunal tissues contribute to
soil organic matter, the main source of organic matter added
to soil is plant litter.
In forests the principal sources
are the leaves, stems, branches, roots, bark, fruits, and
seeds of trees and shrubs (Lutz and Chandler Jr. 1955).
In
-savanna vegetation, the roots and mature herbaceous portions
of grasses constitute the litter.
The biological effects
of the plant litter includes supply of an energy source for
soil organisms and liberation of nutrients for higher plants
( ~ u t zand Chandler Jr. 1955)-
Thus when plant litter reaches
the soil surface it is rapidly colonized by soil microorganisms
and soil animals which degrade the material to obtain their
food supply.
The major groups of litter-decomposing organisms in
the soil are bacteria, actinomycctes, fungi, protozoa, nematodes,
enchytraeid and lumbricid worms, and some insects or their larvae
(Dickinson, 1974).
Fresh plant litter in first acted upon by soil
animals (earthworms, termites, beetles, fly larvae, millipedes,
woodlice, etc.) which fragment the refractory material into smaller
particles, thus exposing a greater surface area for microbial attack.
Some of the chopped material passes through the guts of the soil
animals unchanged but the good moisture relationship of the voided
faecal material makes it a suitable substrate for microbial exploitation (~uhnelt,1961).
The microorganisms along with some soil
#
animals secrete carbohydrases into the soil environment which complete
the hydrolysis of the polysaccharide components of the plant litter
(~iss
e t &., 1975).
In the humid tropics the main decomposers of plant litter are
the fungi which are adapted to activity in acid soils (Alexander,
1977). But under anaerobic or near neutral soil conditions bacteria
may play a significant role.
The black rot and white rot fungi
are the main decomposers of wood.
The rate of decomposition of plant litter by soil organisms
depends on its composition.
The organic constituents of plants are
comnonly divided into six broad categories (Allison, 1973; Alexander,
1977):
(a) cellulose
(b)
hemicelluloses
(c)
lignin
-46(d)
water-soluble fraction (simple sugars, m i n o acids,
and aliphatic acids).
(e)
Ether- and alcohol-soluble fraction (fats, oils, waxes,
resins and pigments).
(f)
Proteins.
As the plant ages, the content of water-soluble constituents,
proteins, and minerals decreases, and the percentage abundance of
On
cellulose, hemicelluloses, and lignin rises (Alexander, 1977).
a weight basis, the bulk of the plant is accounted for by cellulose,
the hemicelluloses and lignin (but2 and Chandler Jr.,
1955).
During
the rainy season in the savanna area, there is rapid growth of
pesture and the leaf material contains high amounts of soluble
carbohydrates.
As dry season progresses plants become progressively
lower in proteins, minerals and soluble carbohydrates and higher
in fibre and lignin (~rowder,1974).
When plant litter falls on the
soil surface, starches, simple proteins, cellulose and hemicellulose
constituents are rapidly decomposed by soil organisms while lignins,
fats, waxes and tannins become resistant to decomposition.
In
mature plant tissues, lignin is usually strongly associated with
cellulose and starch to give rigidity to the tissues.
Among the
fungi known to decompos~cellulose are a considerable number of
Ascomvcetes, Basidiomycetes and Fungi imperfecti.
However, the
association with lignin decreases the rate at which cellulose is
decomposed by these organisms (~illiamsand Gray, 1974).
The
resistance of recalcitrant plant constituents stems from their
content of phenolic groups and numerous bond types.
Thus a variety
of microorganisms producing different bond-specific enzymes will be
required to decompose the resistant materials.
However, lignin
undergoes slow decomposition (mainly by fungi) and the modified
lignin combines with proteins to form humus (christman and Oglesby,
1971).
The role of organic matter in soils (store house for plant
nutrients, increase in exchange capacity, increase in water-holding
capacity, increased soil buffering capacity, etc) is attributed to
its humus fraction (Allison, 1973).
The carbon:nitrogen
(c/N) ratio has been used as an index of
b
decomposition (Allison, 1973).
material has a c/N ratio
less
It was estimated that if the plant
than 25, decomposition by micro-
organisms may proceed at the maximum rate possible under the existing
envirorrmental conditions.
However, if the plant material has a c/N
ratio greater than about 25-30 then decomposition may be slowed down
initially unless a little nitrogen fertilizer was added to bring the
C/N ratio below the critical range near 25.
Thus the amount of
available nitrogen frequently becomes a limiting factor in the
decomposition of plant polysaccharides ( ~ u t zand Chandler, Jr. 1955).
The C/N ratio requirement is variable with the microoragnisms.
Fungi
can adapt to higher C/N ratios than bacteria (~lexander,1977).
However, decomposition rates of plant litter based on C/N ratios
were made without due recognition of the role of extracellular polysaccharidases that actually mediate the hydrolysis process.
enzymes could function under conditions unfavourable for soil
These
-48microorganisms ( S k u j i n s , 1976; Kiss e
t
&. ,
1975).
One commonly h e l d view,was t h a t humid t r o p i c a l s o i l s have low
o r g a n i c matter c o n t e n t b e c a u s e o f t h e i r s a n d y
n a t u r e , poor n u t r i e n t
s t a t u s , h i g h t e m p e r a t u r e s a n d h i g h d e c o m p o s i t i o n rates.
But it i s
now known t h a t o r g a n i c matter c o n t e n t o f t r o p i c a l s o i l s i s n o t v e r y
d i f f e r e n t from t h a t i n t h e t e m p e r a t e r e g i o n ( B i r c h a n d F i e n d , 1956).
The r e a s o n why humid t r o p i c a l s o i l s are h i g h e r i n o r g a n i c matter
than g e n e r a l l y believed i s t h e absence of a d i r e c t r e l a t i o n s h i p
between c o l o u r a n d o r g a n i c c a r b o n c o n t e n t .
Highly w e a t h e r e d o x i s o l s
have h i g h e r o r g a n i c m a t t e r cont,ents t h a n t h e i r r e d d i s h c o l o u r would
i n d i c a t e (Sanchez, 1976).
The a n n u a l a d d i t i o n o f f r e s h o r g a n i c matter as l i t t e r , b r a n c h e s
and dead r o o t s i s o f t h e o r d e r o f 5 t o n s / h a o f d r y matter i n t r o p i c a l
I
f o r e s t and about 1 ton/ha
i n t e m p e r a t e f o r e s t s (Sanchez, 1976).
Because o f t h e l a c k o f s i g n i f i c a n t t e m p e r a t u r e o r m o i s t u r e l i m i t a t i o n s
throughout t h e y e a r , t h e r a t e of o r g a n i c m a t t e r decomposition i s f i v e
t i m e s greater in
udic t r o p i c a l f o r e s t s than i n temperate f o r e s t s .
C o n s e q u e n t l y , e q u i l i b r i u m o r g a n i c m a t t e r c o n t e n t s a r e s i m i l a r (Sanchez
a n d Buol, 1 9 7 5 ) .
T r o p i c a l s a v a n n a s add from 0.5 t o
l i t t e r t o s o i l (.Sanchez,
1976).
1.5 t o n s / h a o f
Humid t r o p i c a l f o r e s t s show a marked
o r g a n i c matter a c c w n u l a t i o n i n t h e t o p s o i l a s a r e s u l t o f l i t t e r
f a l l a n d t h e f a s t e r growth r a t e o f t h e f o r e s t trees.
Fresh organic
m a t t e r a d d i t i o n s i n g r a s s l a n d a r e p r i m a r i l y i n t h e form o f r o o t
decomposition.
The low l e v e l s o f o r g a n i c m a t t e r i n savanna s o i l s
arise from t h e i r p r e d o m i n a n t l y sandy n a t u r e , t h e aluminium t o x i c i t y
I
-49problems in the subsoils, and the constant burning of the grass
(Jones, 1973).
The organic matter level is usually higher in undisturbed
soils because all native vegetation remains on the soil, erosion
is negligible and oxidation is at a minimum.
Thus the cutting down
of forest trees for fannland drastically reduces annual additions
of organic matter to soil.
In the savanna, exposure and ploughing
result in four-fold increases in decomposition rate
o rams,
1971).
Shifting cultivation, however, seldom results in substantial soil
organic matter depletion.
But when overpopulation narrows the crop:
b
fallow ratio, organic carbon content drops (Greenland and Nye, 1959).
It has been suggested (Jones, 1973) that the length of rainy
season is more important in determining organic matter status and
quality than annual rainfall, and that poor drainage might be
considered as effectively prolonging the wet season.
The organic
matter content of humid tropical forest soils is higher during the
dry season than the r ~ i n yseaFon because of relatively low litter
fall and higher decomposition rates in the wet season ( ~ e n n&~.
a.,1949).
Agbim (1987) showed that the decomposition of litter
from some West African plant species was limited by dry season
moisture stress.
The influence of parent material and altitude on organic matter
tatu us are interpreted in the context of geographically limited areas.
Soils developed on shales tend to contain, by virtue of their high
c l a y c o n t e n t , l a r g e amounts o f o r g a n i c m a t t e r t h a n t h e sandy
s o i l s developed on g r a n i t e s a n d s a n d s t o n e s .
t h e well-drained
However, most o f
s o i l s o f t h e savanna r e g i o n s o f W e s t A f r i c a
a r e formed on a c i d basement complex r o c k s ( g r a n i t e s , g n e i s )
a n d s a n d s t o n e s (Sanchez, 1 9 7 6 ) .
4.
Forest-Savanna Mosaic V e g e t a t i o n Cover
F o r e s t - s a v a n n a mosaic i s a v e g e t a t i o n c o v e r i n which
t h e humid t r o p i c a l r a i n f o r e s t a n d a d e r i v e d s a v a n n a e x i s t
b
i n t h e same l o c a t i o n ( ~ e a y , 1959).
I n t h e s e mosaic areas,
t h e f o r e s t s a r e i n most c a s e s t h i c k w h i l e t h e a d j a c e n t s o i l
c o v e r i s p r e d o m i n a n t l y g r a s s w i t h a few s c a t t e r e d young trees.
The f o r e s t a n d t h e savanna grow on t h e s a m e p a r e n t material
-
false-bedded sandstone o r unconsolidated sedimentary
materials (Akamigbo and Asadu,
1983; Unamba-Oparah,
1987b)
a n d r e c e i v e t h e same amount o f r a i n f a l l a n d s u n s h i n e i n t e n s i t y ,
t h a t i s , have t h e s a m e t y p e o f c l i m a t e .
Thus n a t u r a l l y one
s h o u l d e x p e c t t h e v e g e t a t i o n t o be homogenous
-
a thick
t r o p i c a l r a i n f o r e s t b e c a u s e o f t h e humid t r o p i c a l c l i m a t e .
-51The f o r e s t - s a v a n n a t y p e o f v e g e t a t i o n i s commonly o b s e r v e d i n most
p a r t s o f s o u t h e a s t e r n N i g e r i a (Keay, 1959; I g b o z u r i k e , 1975) where
w i t h i n a few m e t e r s , one c a n pass from a c l o s e d f o r e s t t o an a l m o s t
open g r a s s l a n d .
I n some p l a c e s t h e t h i c k f o r e s t a n d g r a s s l a n d are
s e p a r a t e d by a s h r u b l a n d c h a r a c t e r i s e d by s m a l l trees a n d t a l l
grasses.
The s h r u b l a n d r e p r e s e n t s a s a v a n n a zone t h a t h a s n o t been
A s t h e p r e s s u r e on t h e l a n d i n c r e a s e s
h e a v i l y c r o p p e d f o r many y e a r s .
f o r farmland, t h e shrubland e v e n t u a l l y d i s a p p e a r s and t h e d e r i v e d
savanna ( g r a s s l a n d ) g r a d u a l l y e n c r o a c h e s i n t o t h e humid t r o p i c a l
rainforest,
b
V e g e t a t i o n as a b i o t i c f a c t o r , s i g n i f i c a n t l y i n f l u e n c e s t h e
c h a r a c t e r i s t i c s of t h e s o i l .
of n u t r i e n t - d e m a n d i n g
Eyre ( 1 9 6 7 ) n o t e d t h a t w i t h o u t a c o v e r
v e g e t a t i o n , no s o i l c o u l d m a i n t a i n i n d e f i n i t e l y
a h i g h b a s e s t a t u s i n a humid c l i m a t e , no m a t t e r how r i c h i n b a s e s
t h e u n d e r l y i n g p a r e n t m a t e r i a l may be.
The d i f f e r e n c e s i n t h e
c h a r a c t e r i s t i c s of t h e s o i l s i n t h e s a v a n n a and f o r e s t e c o t o n e l i e
b a s i c a l l y on t h e t y p e o f v e g e t a t i o n c o v e r , s i n c e t h e y s h a r e t h e same
p a r e n t r o c k a n d e x p e r i e n c e t h e same c l i m a t e .
S e v e r a l w o r k e r s (Ojanuga, 1980; Osakwe, 1981; and Unamba-Oparah,
1987b) have s t u d i e d t h e c h a r a c t e r i s t i c s of t h e f o r e s t - s a v a n n a mosaic
s o i l s i n Nigeria.
These s t u d i e s i n d i c a t e t h a t when f o r e s t i s c l e a r e d
t o g i v e rise t o a savanna t y p e o f v e g e t a t i o n , t h e r e i s l o s s i n s o i l
nutrient s t a t u s , decrease i n s o i l organic matter content, increase i n
sanciy t e x t u r e o f s o i l a n d a n i n c r e a s e i n s u s c e p t i b i l i t y o f t h e s o i l s
-52t o e r o s i o n hazards.
These s t u d i e s show how t h e human f a c t o r
a f f e c t s s o i l chemical and p h y s i c a l c h a r a c t e r i s t i c s .
However,
l i t t l e o r no a t t e n t i o n h a s been g i v e n t o assess t h e e f f e c t s
o f human a c t i v i t y on t h e b i o c h e m i c a l c h a r a c t e r i s t i c s o f t h e s e
soils.
A s t u d y o f s o i l polysaccharidases could give
an
i n d i c a t i o n o f t h e rate o f c a r b o n c y c l i n g and m i n e r a l i z a t i o n o f
o r g a n i c matter i n f o r e s t - s a v a n n a mosaic s o i l s .
5.
Methodology o f S o i l Enzyme Measurement
S o i l enzymology is s t i l l a n e v o l v i n g d i s c i p l i n e , t h u s
methods o f s o i l enzyme a s s a y s are y e t t o s t a b i l i z e .
T h e r e are
no s t a n d a r d p r o c e d u r e s f o r s o i l enzyme measurement.
llMethods i n
Enzymology1' (Academic Press) h a s now r e a c h e d o v e r o n e hundred
and t h i r t y - f i v e
volumes b u t v e r y few a r t i c l e s d e a l , even s u p e r -
f i c i a l l y , K i t h s o i l enzyme measurements.
Not s u r p r i s i n g l y
t h e r e f o r e , t h e m a j o r r e s e a r c h g r o u p s have e i t h e r developed t h e i r
own a s s a y s from f i r s t p r i n c i p l e s o r r a d i c a l l y a d a p t e d t h o s e
a l r e a d y published.
Some o f t h e m e t h o d o l o g i e s f o r s o i l enzyme
a s s a y s h a v e been r e p o r t e d by Roberge (1978) a n d T a b a t a b a i (1982).
The d i s p a r i t y i n t h e methods a d o p t e d i n d i f f e r e n t p l a c e s are i n
part a r e f l e c t i o n o f t h e h e t e r o g e n e i t y o f t h e s o i l e n v i r o n m e n t ;
a n environment which h a s n o t o n l y t h e o b v i o u s m i n e r a l o g i c a l
b i o l o g i c a l v a r i a t i o n s b u t a l s o some a p p a r e n t l y less e a s i l y
d e f i n e d g e o g r a p h i c a l o n e s a s w e l l ( ~ u r n s , 1978).
and
I n c l a s s i c a l enzymology, e x t r a c t e d and p u r i f i e d enzymes a r e
a s s a y e d i n t h e p r e s e n c e of e x c e s s s u b s t r a t e d i s s o l v e d i n a b u f f e r
and t h e r e a c t i o n mixture i s a g i t a t e d f o r a f i x e d p e r i o d of t i m e .
M t h t h e pH of t h e r e a c t i o n and t h e t e m p e r a t u r e of i n c u b a t i o n a r e
t h e optimum f o r a c t i v i t y .
These c o n d i t i o n s g i v e t h e maximum p o t e n t i a l
c a t a l y t i c a c t i v i t y ( ~ u r n s ,1978).
There i s , of c o u r s e , a world of
d i f f e r e n c e between p u r e enzymology and t h e i n v i v o a c t i v i t y of s o i l
enzymes.
Because of t h e v e r y s t r o n g s o r p t i v e and o t h e r chemical
i n t e r a c t i o n s between s o i l enzyme p r o t e i n s and o r g a n i c a s w e l l a s
inorganic s o i l constituents,
i t b a s been e x t r e m e l y d i f f i c u l t t o
e x t r a c t a c t i v e enzymes from t h e s o i l .
Thus measurements a r e i n d i r e c t .
Often c r u d e s o i l s u s p e n s i o n s a r e used i n s o i l enzyme a s s a y s .
It i s
argued t h a t e x t r a c t i o n of pure s o i l enzymes may y i e l d o n l y f r a c t i o n a l
amounts and might d e c r e a s e a c t i v i t y s i n c e t h e o r g a n i c and i n o r g a n i c
s o i l p a r t i c l e s are n e c e s s a r y t o s t a b i l i z e s o i l enzymes and p r e v e n t
t h e i r i n h i b i t i o n o r i n a c t i v a t i o n ( S k u j i n s , 1976).
Secondly, s o i l
enzymes are r a r e l y p r e s e n t e d w i t h e x c e s s s u b s t r a t e ; w i t h i n t h e s o i l
environment, t h e m o i s t u r e l e v e l i s extremely v a r i a b l e and w h i l s t
s o i l s do have a s i g n i f i c a n t b u f f e r i n g c a p a c i t y i t is c e r t a i n l y n o t
up t o t h e s t a n d a r d o f , s a y 0.5 M phosphate o r t r i s - H C l
1978).
buffer ( ~ u r n s ,
Temperature and pH measurements a r e o f t e n n e c e s s a r y t o
determine c o n d i t i o n s f o r optimum enzyme a c t i v i t y .
and t e m p e r a t u r e a r e a l l macro-environment
(i.e.
However, s o i l pH
bulk s o i l ) measurements
and may n o t be synonymous w i t h t h o s e i n t h e molecular s i z e zone where
t h e enzyme and i t s s u b s t r a t e i n t e r a c t , t h a t i s , t h e s o i l p a r t i c l e
surfaces ~ ( ~ u r n s1978).
,
A
prime problem in soil enzymology has been finding an ideal
sterilizing agent to facilitate the separation of extracellular
enzyme activities from those of microorganisms.
The distinction
between extracellular enzyme activity and activity associated with
intact microbial cells requires that the technique should
(a)
preserve the stability of the cell membrane against
liberation of intracellular enzymes or assimilation of
products or substrates;
b
(b)
prevent further microbial synthesis of the enzyme; and
(c)
prevent inactivation of the activity being measured
(Skujins, 1967).
Despite these problems, individual soil enzymes are more readily
detected than other organic substances in soil because small quantities
effect measurable changes in added substrate.
,
At best, activities of
soil enzymes are measured under standardized conditions.
Nevertheless,
assay conditions are chosen arbitrarily or based on experience, and differ
markedly from those of the natural soil environment ( ~ o b e r ~ e1978).
,
quently,soils are incubated as
buffered suspensions with natural
or synthetic substrate in the presence of a sterilizing agent, for
a period of time and the product(s) or the residual substrate is
measured in the filtered suspension.
Usually, because of kinetic
considerations, the substrate5 are a d d e d in abnormally high concentrations (Roberye, 1978).
The accuracy of any assessment of soil enzyme activity is
I
1
fie-
-55c o n s i d e r a b l y i n f l u e n c e d by c o n d i t i o n s d u r i n g s t o r a g e o f s o i l a f t e r
i t s c o l l e c t i o n , m e t h o d s o f p r e p a r i n g s a m p l e p r i o r t o enzyme m e a s u r e ment a n d e x t r a c t i o n , c o n d i t i o n s d u r i n g t h e a c t u a l enzyme d e t e r m i n a t i o n
and procedures u s e d f o r a n a l y s i s o f t h e s u b s t r a t e s and t h e p r o d u c t s
( ~ o b e r ~ e1 9
, 78).
The f o l l o w i n g c o n s i d e r a t i o n s a r e made d u r i n g s o i l enzyme
assays
(Burns, 1978; R o b e r g e , 1 9 7 8 ) :
L a r g e s i z e s o i l p a r t i c l e s may i n c r e a s e c e r t a i n
enzymatic a c t i v i t i e s and d e c r e a s e o t h e r s , b u t t h e
g e n e r a l l y u s e d 2 mm s i e v e f o r m i n e r a l s o i l a n d 4 mm
b
s i e v e f o r organic appear adequate.
Shaking exposes
a l i t t l e more crumb m a t r i x enzyme t o s u b s t r a t e h e n c e
i n c r e a s e s enzyme a f f i n i t y t o s u b s t r a t e .
E n z y m a t i c a c t i v i t i e s a r e d e c r e a s e d by s o i l s t o r a g e
(Ross, 1%5),
more i f t h e s o i l i s i n a n a i r - d r y s t a t e
than i n a m o i s t s t a t e , a n d more a t room t e m p e r a t u r e
0
o r f r o z e n t h a n a t 5 C.
P a n c h o l y a n d R i c e ( 1 9 7 2 ) , showed
that a m y l a s e a c t i v i t y was r e d u c e d w h e t h e r s o i l was s t o r e d
a i r - d r i e d o r m o i s t , a t room t e m p e r a t u r e , a t
0
-20 C.
~ O C
o
Thus f r e s h s o i l s a m p l e s s h o u l d b e u s e d .
r at
Also
s o i l s s h o u l d be s i e v e d i n t h e f i e l d - m o i s t c o n d i t i o n .
Maximum e n z y m a t i c a c t i v i t i e s o f t e n o c c u r i n b u f f e r e d
s o i l a t a pH a r o u n d
7.0.
They t e n d t o o c c u r a t a l o w e r
pH when t h e o r i g i n a l pH o f t h e s o i l i s a c i d a n d a t a
h i g h e r pH when t h e o r i g i n a l pH o f t h e s o i l i s a l k a l i n e .
-56The pH o f t h e b u f f e r s h o u l d be a s c l o s e t o t h e o r i g i n a l
s o i l pH a s p o s s i b l e .
B u f f e r s perform e i t h e r one o r
both o f t h e i r p r i n c i p a l f u n c t i o n s during a n assay: t h e y
a d j u s t t h e pH t o t h a t r e q u i r e d f o r optimum a c t i v i t y
a n d m a i n t a i n t h a t pH f o r t h e d u r a t i o n o f t h e r e a c t i o n ,
i.e.,
t h e y e n s u r e t h a t t h e r e i s no s i g n i f i c a n t pH
s h i f t during t h e reaction.
However, some b u f f e r s are
known t o d e c r e a s e t h e a c t i v i t y of some s o i l enzymes,
hence d i s t i l l e d w a t e r s h o u l d b e u s e d i n p l a c e .
Radulescu (1972), r e p p r t e d t h a t a m y l a s e a c t i v i t y was
lower i n r e a c t i o n m i x t u r e s b u f f e r e d w i t h acetic a c i d sodium a c e t a t e s o l u t i o n a t pH 5.6
than i n mixtures i n
which d i s t i l l e d w a t e r was used i n s t e a d o f b u f f e r .
( i v ) Accumulated ( e x t r a c e l l u l a r ) enzymes a r e a s s a y e d u n d e r
c o n d i t i o n s which minimize o r e l i m i n a t e m i c r o b i a l
p r o l i f e r a t i o n , e i t h e r by u s i n g s e n s i t i v e a s s a y s w i t h
b r i e f i n c u b a t i o n p e r i o d s , o r by u s i n g s t e r i l i z e d
a n d / o r i r r a d i a t e d s o i l samples.
An i d e a l s t e r i l i z a t i o n
a g e n t f o r e x t r a c e l l u l a r enzyme d e t e c t i o n i n s o i l would be
one t h a t would c o m p l e t e l y i n h i b i t a l l m i c r o b i a l a c t i v i t i e s
t o p r e v e n t f u r t h e r m i c r o b i a l s y n t h e s i s o f t h e enzyme,
p r e s e r v e t h e s t a b i l i t y of t h e m i c r o b i a l c e l l membranes
a g a i n s t l i b e r a t i o n o f i n t r a c e l l u l a r enzymes o r a s s i m i l a t i o n
of s u b s t r a t e s a n d / o r p r o d u c t s , a n d would a l s o n o t
i n t e r f e r e w i t h t h e a c t i v i t y o f t h e enzyme a s s a y e d
S k u j i n s , 1967).
Although s o i l i r r a d i a t i o n t e c h n i q u e s have been
u s e d ( ~ c L a r e n & t d.,1 % ~ )t o l u e n e i s t h e u n i v e r s a l a g e n t u s e d
i n s o i l enzyme a s s a y s b e c a u s e o f i t s e f f e c t i v e b i o s t a t i c c h a r a c t e r
p r e v e n t i n g namely, t h e s y n t h e s i s o f enzymes, t h e p r o l i f e r a t i o n o f
m i c r o o r g a n i s m s , and t h e a s s i m i l a t i o n o f r e a c t i o n s u b s t r a t e s and
p r o d u c t s ( ~ e c ka n d P o s c h e n r i e d e r , 1963; F r a n k e n b e r g e r J r . and
Johanson, 1986).
Toluene i s however i n e f f e c t i v e d u r i n g l o n g
i n c u b a t i o n p e r i o d s e x c e e d i n g 48 h o u r s .
-
MATERIALS AND METHODS
V e g e t a t i o n Of S t u d y Area
The area o f s t u d y w a s a t Owerre Eze Orba v i l l a g e , s i t u a t e d
east o f t h e U n i v e r s i t y o f N i g e r i a Nsukka farm.
This site w a s
s e l e c t e d b e c a u s e t h e v e g e t a t i o n c o v e r formed a t y p i c a l f o r e s t s a v a n n a m o s a i c ; a t h i c k humid t r o p i c a l f o r e s t , s h r u b l a n d a n d g r a s s l a n d e x i s t e d o n t h e same' l o c a t i o n . hence i t was assumed t h a t t h e i r
s o i l s o r i g i n a t e d from t h e s a m e p a r e n t material
Nsukka which i s t h e f a l s e - b e d d e d s a n d s t o n e .
-
t h e parent rock of
Secondly, t h e l o c a t i o n
w a s n e a r t h e F a c u l t y o f A g r i c u l t u r e , U n i v e r s i t y o f N i g e r i a , Nsukka
*
s o t h a t f r e s h s o i l s a m p l e s c o u l d q u i c k l y b e b r o u g h t back t o t h e
l a b o r a t o r y f o r immediate a n a l y s i s f o r e n z y m a t i c a c t i v i t y .
The s a v a n n a e c o t o n e ( i . e .
s h r u b l a n d and g r a s s l a n d ) occupied a
v e r y e x t e n s i v e area a n d c o n s t i t u t e d t h e main v e g e t a t i o n c o v e r , w h i l s t
t h e f o r e s t v e g e t a t i o n o c c u p i e d j u s t a b o u t 100 h e c t a r e s o f l a n d .
The
f o r e s t v e g e t a t i o n c o v e r h a d no c r o p p i n g h i s t o r y , t h a t i s , i t h a s b e e n
a virgin forest.
I t c o n s i s t e d of heterogeneous g i a n t t r e e s p e c i e s
w i t h v e r y p o o r canopy undergrowth.
Among t h e t r e e s p e c i e s w e r e
C h l o r o p h e r a e x c e l s a , S t e r c u l i a c e a e , Moraceae, Ulmaceae a n d a h o s t
of other unidentifieii
species.
During t h e d r y s e a s o n t h e r e was
h i g h a c c u m u l a t i o n o f l i t t e r on t h e f o r e s t f l o o r , a r i s i n g m a i n l y from
leaf f a l l ,
The s a v a n n a p o r t i o n c o n s i s t e d o f h e r b a c e o u s f i r e - t e n d e r g r a s s
s p e c i e s ( I m p e r a t a c y l i n d r i c a , Androponon, P h y l l a n t h i s d i s c o i d e u s , e t c , )
a n d f i r e - t e n d e r young t r e e s p e c i e s ( L k m i e l l a o l i v e r i , A f z e l i a
africana, etc.),
there.
a n d some palm a n d cashew trees s c a t t e r e d h e r e a n d
The s h r u b l a n d a r e a h a s been l e f t f a l l o w f o r t h e p a s t f i v e
y e a r s w h i l e t h e g r a s s l a n d a r e a h a s n o t been c r o p p e d f o r t h e p a s t
two y e a r s , a l t h o u g h b o t h a r e a s have undergone c y c l e s o f bush b u r n i n g
i n t h e p r e v i o u s dry s e a s o n s .
Both s h r u b l a n d a n d g r a s s l a n d a r e a s had
no h i s t o r y o f f e r t i l i z e r a p p l i c a t i o n and w e r e l e f t f a l l o w t h r o u g h o u t
t h e p e r i o d o f s t u d y (Feb.'87
Low-domed,
-
Jan.'88).
capped o r mushroom-shaped
t e r m i t e mounds ( c h a r a c t e -
r i s t i c o f Nasute t e r m i t e s ) w e r e o b s e r v e d i n t h e t h r e e v e g e t a t i o n
t y p e s , b u t w e r e p a r t i c u l a r l y abundant i n t h e f o r e s t v e g e t a t i o n .
b
Earthworm castes w e r e a l s o p r e s e n t on t h e s o i l s u r f a c e o f t h e t h r e e
v e g e t a t i o n z o n e s ; t h e y w e r e more a b u n d a n t i n t h e r a i n y s e a s o n t h a n
during t h e dry season.
S o i l .samples w e r e c o l l e c t e d w i t h i n a b o u t o n e hundred s q u a r e
meters from e a c h o f t h e t h r e e v e g e t a t i o n t y p e s ( f o r e s t , s h r u b l a n d
and g r a s s l a n d ) e a c h month f o r t w e l v e months ( ~ e b . ' 8 7
-
Jan.'88)
and
a n a l y s e d f o r a m y l a s e a n d c e l l u l a s e a c t i v i t i e s , a n d f o r some chemical
and p h y s i c a l p r o p e r t i e s .
A t t h e e n d o f t h e s t u d y ( J a n u a r y 1988) m a t u r e l e a f l i t t e r was
c o l l e c t e d from t h e f l o o r o f e a c h o f t h e t h r e e v e g e t a t i o n t y p e s a n d
a n a l y s e d f o r s t a r c h , c e l l u l o s e and l i g n i n c o n t e n t .
R a i n f a l l d a t a f o r Nsukka was o b t a i n e d from t h e M e t e o r o l o g i c a l
S t a t i o n , o f t h e F a c u l t y of A g r i c u l t u r e , U n i v e r s i t y o f N i g e r i a ,
1.
Soil Sampling
S o i l samples ( 0
-
15 c m d e p t h ) were c o l l e c t e d between 1 9 t h a n d
22nd d a y o f e a c h month ( ~ e b . ' 8 7 - J a n . ' 8 8 )
from e a c h o f t h e t h r e e
v e g e t a t i o n t y p e s u s i n g a hand a u g e r o f 2 cm c o r e d i a m e t e r .
The
s a m p l e s w e r e randomly t a k e n from f i f t e e n s i t e s w i t h i n e a c h v e g e t a t i o n
cover.
S i x c o r e s w e r e dug a t e a c h s i t e a n d t h e n t h e c o r e s from
f i v e a d j a c e n t sites bulked t o g i v e one of t h e t h r e e samples i n a
p a r t i c u l a r v e g e t a t i o n c o v e r f o r s e p a r a t e a n a l y s i s f o r s o i l enzyme
activity.
The s a m p l e s c o l l e c t e d a t e a c h p e r i o d were i m m e d i a t e l y
b r o u g h t t o t h e l a b o r a t o r y a n d a n a l y s e d f o r enzyme a c t i v i t y .
#
2.
S o i l Analysis
S o i l s a m p l e s f o r c h e m i c a l and p h y s i c a l a n a l y s i s w e r e a i r - d r i e d
and p a s s e d t h r o u g h a 2 mm s i e v e .
,
A l l d e t e r m i n a t i o n s were i n d u p l i c a t e
a n d e x c e p t f o r p a r t i c l e s i z e a n a l y s i s , a l l o t h e r d e t e r m i n a t i o n s were
I
p e r f o r m e d e a c h month t h r o u g h o u t t h e c o u r s e o f s t u d y .
I
P a r t i c l e s i z e a n a l y s i s w a s p e r f o r m e d by t h e Houyoucos
( 1 9 3 6 ) h y d r o m e t e r method u s i n g sodium h e x a m e t a p h o s p h a t e
( c a l g o n ) as d i s p e r s i n g a g e n t .
S o i l pH was d e t e r m i n e d i n a s o i l s u s p e n s i o n w i t h a
soil/water a n d s o i l / O . l N
KC1 r a t i o o f 1:2.5,
using
a Beckman pH m e t e r .
E x c h a n g e a b l e c a t i o n s werp e x t r a c t e d w i t h n e u t r a l
normal amnonium a c e t a t e b u f f e r ( P e e c h
& aJ.,
19It7)
E x c h a n g e a b l e c a l c i u m a n d magnesium w e r e t h e n d e t e r m i n e d
-61by V e r s e n a t e t i t r n t i o n , and e x c h a n g e a b l e p o t a s s i u m
and sodium by f l a m e photometry.
(d)
C a t i o n exchange c a p a c i t y (C.E.C)
by washing the NH4-saturated
w a s determined
s o i l sample w i t h methanol
t o f r e e i t o f excess amnonium a c e t a t e .
The amnonium
was f u r t h e r e x t r a c t e d w i t h 1.ON KC1 and steam
d i s t i l l e d orlt w i t h MgO i n t o 4% b o r i c a c i d s o l u t i o n .
The amnoniun (NH')
4
was t h e n d e t e r m i n e d by t i t r a t i o n
w i t h s t a n d a r d HC1.
(e)
b
O r g a n i c m a t t e r was d e t e r m i n e d by t h e chromic a c i d
o x i d a t i o n method o f Walkley and Black (1934) a s m o d i f i e d
by Smith and Weldon ( 1 9 4 0 ) .
(f)
T o t a l n i t r o g e n was d e t e r m i n e d by k j e l d a h l d i g e s t i o n
u s i n g c o n c e n t r a t e d s u l p h u r i c a c i d a n d a sodium
sulphate-copper s u l p h a t e c a t a l y s t mixture.
Ammonia
i n t h e d i g e s t was d i s p l a c e d w i t h 45% NaOH s o l u t i o n
and d i s t i l l e d i n t o 4% b o r i c a c i d and t h e n d e t e r m i n e d
by t i t r a t i o n w i t h 0.05N
(g)
HCl.
A v a i l a b l e phosphorus w a s d e t e r m i n e d by B r a y ' s method I1
(Bray and K u r t z , 1945).
A i r - d r y s o i l was e x t r a c t e d
w i t h B r a y t s s o l u t i o n a n d f i l t e r e d t h r o u g h a Whatman No.
f i l t e r paper.
The amount o f a v a i l a b l e phosphorus
i n t h e e x t r a c t was t h e n d e t e r m i n e d by development o f
42
-62a molybdenum b l u e c o l o u r a n d e x t r a p o l a t i o n o f t h e
r e a d i n g o f t h e c o l o r i m e t e r from a p r e v i o u s l y
prepared s t a n d a r d curve.
(h)
S o i l mixture was d e t e r m i n e d by w e i g h i n g f r e s h s o i l
samples i n a g l a s s c r u c i b l e and drying
0
i n a n oven a t 105 C t o c o n s t a n t w e i g h t .
the soil
Results
were g i v e n as p e r c e n t a g e m o i s t u r e c o n t e n t o n oven
dry basis.
3.
S o i l Enzyme Assay
b
S o i l s a m p l e s c o l l e c t e d e a c h month ( ~ e b . ' 8 7 - J a n . ' 8 8 )
were
placed i n p l a s t i c t u b e s and immediately brought t o t h e l a b o r a t o r y
f o r enzyme s t u d i e s , t o a v o i d a i r - d r y i n g .
The s o i l s w e r e t h e n s i e v e d
i n t h e f i e l d m o i s t c o n d i t i o n u s i n g a 2 mm s i e v e .
r o o t s w e r e removed by h a n d - p i c k i n g .
I
Visible plant
Enzyme a c t i v i t i e s w e r e measured
o n t r i p l i c a t e s a m p l e s o f f r e s h s o i l by i n c u b a t i n g a s o l u t i o n o f s t a r c h
w i t h d i s t i l l e d w a t e r ( f o r a m y l a s e a s s a y ) o r CMC w i t h a c e t a t e b u f f e r
( f o r cellulase assay).
,
Toluene w a s used t o i n h i b i t m i c r o b i a l
growth d u r i n g t h e i n c u b a t i o n o f t h e s o i l s u s p e n s i o n w i t h s u b s t r a t e s .
F o r c o n v e n i e n c e , t h e enzymes h y d r o l y s i n g s t a r c h a n d c e l l u l o s e h a v e
been c a l l e d a m y l a s e a n d c e l l u l a s e r e s p e c t i v e l y , b u t t h e e x a c t
n a t u r e o f t h e s e enzymes h a s n o t been f u l l y e l u c i d a t e d .
The method f o r a m y l a s e d e t e r m i n a t i o n i n s o i l was a d a p t e d from
Kelw (19751, a n d t h a t f o r c e l l u l a s e a c t i v i t y from P a n c h o l y a n d
I
- 63Rice (1973a).
A l l d a t a w e r e a n a l y s e d u s i n g t h e s t a t i s t i c a l methods
o f L i t t l e a n d H i l l s (1978) and S t e e l and T o r r i e (1980).
The data
w e r e a r r a n g e d t o f i t i n t o a r a n d o m i z e d c o m p l e t e b l o c k (RCB) d e s i g n
where t h e t h r e e v e g e t a t i o n t y p e s r e p r e s e n t e d t h e t r e a t m e n t s a n d
months
t h e blocks.
A l l v a l u e s w e r e compared u s i n g F i s h e r ' s L e a s t
S i g n i f i c a n t D i f f e r e n c e (F-LSD).
(a)
Assay o f S o i l Amylase A c t i v i t y
To d e t e r m i n e a m y l a s e a c t i v i t y ( K e l l y ,
19751, 5 g o f
sieved f r e s h soil (field-moibt condition) w a s placed i n a
50 m l E r l e m e y e r f l a s k a n d 1.5 m l o f t o l u e n e w a s a d d e d ; t h e
m i x t u r e w a s s h a k e n a n d a l l o w e d t o s t a n d f o r 25 m i n u t e s .
t h i s w a s a d d e d 10 m l o f d i s t i l l e d w a t e r a n d 5 m l o f 2%
solution of soluble starch.
To
(w/v)
The f l a s k was t h e n s t o p p e r e d
and p l a c e d i n a n i n c u b a t o r (water-bath w i t h thermometer)
0
a t 37 C f o r 5 h o u r s .
A f t e r i n c u b a t i o n 15 m l o f d i s t i l l e d
w a t e r was a d d e d , t h e f l a s k s h a k e n , a n d
t h e suspension w a s centrifuged a t
produce a c l e a r s u p e r n a t a n t .
R
10 m l a l i q u o t o f
3500 rpm f o r 20 m i n u t e s t o
One m i l l i l i t r e o f t h e c l e a r
s u p e r n a t a n t w a s t h e n a n a l v s e d f o r r e d u c i n g s u g a r s by a
m o d i f i e d Nelson-Somogyi
method ( N e l s o n , 1 9 6 4 ) .
Controls
c o n s i s t i n g o f s o i l w i t h o u t s o l u b l e s t a r c h ( s u l ~ s t r n t eb l a n k ) nncl
a u t o c l avcd ~ ol iw i t h s o l n b l r s t a r c h (cn7,y1nc b l a n k ) wcre r u n c o n c u r r e n t l y .
Amylase a c t i v i t y w a s expressed as r e d u c i n g s u g a r s
-64released (in milligrams of glucose equivalents) per gram
of oven dry soil when a mixture of the composition given
0
above was incubated for 5 hours at 37 C.
-(b) Assay of Soil Cellulase Activity
For determination of cellulase activity (Pancholy and
Rice, 1973a)
5 g of sieved fresh soil (field-moist condition)
were placed in a
added.
50 ml Erlenmeyer flask and 0.5 ml of toluene
The contents were mixed thoroughly and after 15
minutes, 10 ml of 0.24 acetate buffer (PH 4.8) were added
b
followed by 10 ml of 1% (w/v) carboxymethylcellulose (CMC).
The flask was then incubated in a water bath for 24 hours
at
30°c.
50 ml of
At the end of the incubation period
distilled water was added, the suspension filtered through
Whatman No.
30 filter paper, and the volume of the filtrate
was made up to 1 0 0 ml with distilled water.
The reducing
sugar content of the filtrate was determined by Nelson's
(1961,) method.
Controls consisting of soil without carboxy-
methylcellulose and autoclaved soil with carboxymethylcellulose were run concurrentlv.
Cellulase activity was expressed as reducing
groups
released (in milligrams of glucose equivalents) per gram of
oven drv soil when a r*eactionmixture of the composition
0
given above was incubated for 2 h hours at 30 C.
-654.
Chemical C o m p o s i t i o n o f P l a n t L i t t e r
F a l l e n l e a f l i t t e r c o l l e c t e d from t h e canopy o f e a c h o f t h e
v e g e t a t i o n t y p e s w a s ground and a n a l y s e d f o r s t a r c h , c e l l u l o s e and
lignin content.
The c a r b o h y d r a t e t o l i g n i n r a t i o f o r e a c h o f t h e
t h r e e l i t t e r samples w a s t h e n c a l c u l a t e d .
A l l determinations w e r e
i n duplicate.
(a)
Cellulose Determination
The method r e p o r t e d by Crampton a n d Maynard ( 1 9 3 8 )
w a s used.
Ground p l a n t l i t t e r w a s d i g e s t e d w i t h g l a c i a
b
a c e t i c a c i d which d i s s o l v e s a l l o t h e r c o n s t i t u e n t s l i k e
protein,
disaccharides,
f a t t y acids, etc.,
c e l l u l o s e and mineral contents unaffected.
but leaves
The r e s i d u e
w a s a c i d i f i e d w i t h conc. n i t r i c a c i d a n d f i l t e r e d through
a gooch c r u c i b l e w i t h a s b e s t o s pad.
The r e s i d u e w a s
t h e n washed w i t h h o t w a t e r , a l c o h o l , b e n z e n e a n d a l c o h o l
i n t h a t order.
A l c o h o l removed w a t e r w h i l e b e n z e n e
removed p i g m e n t s a n d l i p i d s .
0
The r e s i d u e w a s d r i e d a t
1 0 0 C f o r 10 m i n u t e s a n d weighed.
The d r i e d r e s i d u e w a s
t h e n p l a c e d i n a c r u c i b l e and ashed i n a muffle furnace
a t 5 5 0 ~f o~r h a l f a n h o u r .
The d i f f e r e n c e between t h e
d r i e d weight b e f o r e a s h i n g and weight a f t e r a a h i n g gave
t h e c e l l u l o s e c o n t e n t o f t h e p l a n t l i t t e r sample.
r e s u l t s w e r e e x p r e s s e d as g r a m s p e r 1 0 0 grams.
The
(b)
Lignin Determination
L i g n i n d e t e r m i n a t i o n i n t h e ground p l a n t l i t t e r s a m p l e s
was by t h e method o f Adams (1965).
It i s b a s e d on t h e removal
o f t h e a s s o c i a t e d p o l y s a c c h a r i d e s by s o l u b i l i z a t i o n a n d
h y d r o l y s i s w i t h s t r o n g m i n e r a l a c i d (72% s u l p h u r i c a c i d ) a n d
weighing o f t h e d r i e d r e s i d u e as l i g n i n .
The r e s u l t s w e r e e x p r e s s e d as grams p e r 100 grams.
(c)
S t a r c h Determination
S o l u b l e s u g a r s w e r e e x t r a c t e d from f i n e l y d i v i d e d ( g r o u n d
l e a f l i t t e r u s i n g a l t e r n a t e l y 2 m l d i s t i l l e d w a t e r a n d 10 m l
8 a ethanol.
0
The r e s i d u e was d r i e d a t 60 C o v e r n i g h t a n d
d i g e s t e d w i t h 15 m l c o n c e n t r a t e d p e r c h l o r i c a c i d ( ~ a i l e y ,1958).
The d i g e s t was f i l t e r e d t h r o u g h a s i n t e r e d g l a s s f u n n e l a n d 1 m l
o f f i l t r a t e was u s e d f o r a n a l y s i s o f r e d u c i n g s u g a r s u s i n g t h e
phenol-sulphuric
t
a c i d method (Dubios e
a.,1956).
The r e d u c i n g
sugar content expressed a s glucose equivalents w a s obtained
from a s t a n d a r d g l u c o s e c u r v e .
The w e i g h t o f t h e g l u c o s e i n
t h e h y d r o l y s a t e w a s c a l c u l a t e d a s s t a r c h by m u l t i p l y i n g by t h e
factor
0.9 a n d e x p r e s s e d a s a p e r c e n t a g e o f t h e d r y matter.
A s e c o n d p o r t i o n o f t h e ground l i t t e r w a s d r i e d o v e r n i g h t i n
t h e oven t o g i v e t h e d r y matter c o n t e n t .
The f a c t o r 0.9
i s b a s e d on t h e a s s u m p t i o n (13ailey, 1958)
t h a t d u r i n g e n z y m a t i c h y d r o l y s i s o f s t a r c h 90% o f i t s g l u c o s e
c o n t e n t are r e l e a s e d .
I
o mo Oo \ om Qo \ qm o9 \ q3 q o q o. c
g d d d d o d c c d c c c
*
G .o . .o . o
o
~
d
6
L
-
w
E
Q
b
X
l
c
o
I n L n Q Q
O
O
O
O
O
O
O
O
O
C
O
C
~
6
sasec
a~qea6ueyaxa p q o j
8
*
\
S g 3 " o " o 8 o o o 3
.
d d d d d d d d d d o o
b b b u ' b I n \ D b b c o
o o q o o o o o q q q q
d d o d d d d d o o o o
v \ a v \ u ' u ' a b v \ a ; r v \ 4 '
? ? ? ? k t a a b ? y t
o o o o o o d d d o o o
- 70RESULTS AND DISCUSSION
E f f e c t of S o i l T e x t u r e F a c t o r on t h e S o i l Enzyme A c t i v i t i e s
The v a l u e s of some o f t h e p h y s i c a l a n d chemical p r o p e r t i e s
of g r a s s l a n d , s h r u b l a n d a n d f o r e s t s o i l s d e t e r m i n e d o v e r a t w e l v e
month p e r i o d ( ~ e b . '87
3, r e s p e c t i v e l y .
-
Jan.
'88) a r e shown i n T a b l e s 1, 2 a n d
P a r t i c l e size a n a l y s i s showed t h a t s o i l u n d e r g r a s s -
l a n d v e g e t a t i o n c o v e r had a sandy loam t e x t u r e w h i l e s h r u b l a n d
a n d f o r e s t s o i l s b o t h had a s a n d y c l a y loam t e x t u r e ( T a b l e s 1, 2 ,
3 and 4).
The c l o s e t e x t u r a l c l a s s e s a n d t h e h i g h sandy n a t u r e o f
a l l t h e s o i l s under t h e t h r e e v e g e t a t i o n t y p e s (Table 4 ) support
t h e i d e a that t h e s e s o i l s o r i g i n a t e d from t h e same p a r e n t material
-
t h e p a r e n t r o c k o f Nsukka which i s t h e f a l s e - b e d d e d s a n d s t o n e
( ~ k a m i g b oand Asadu,
1983; Unamba-Oparah,
1987b) c o n s i s t i n g c h i e f l y
of s e s q u i o x i d e s a n d k a o l i n i t i c c l a y m i n e r a l s ( J u o , 1981).
Because
o f t h e a p p a r e n t s i m i l a r i t y i n t h e c l a y mineralogy o f t h e t h r e e
c o n t i g u o u s s i t e s , t h e c l a y t y p e f a c t o r would t h e r e f o r e n o t b e
expected t o be a n i s s u e i n r e g a r d t o any s i g n i f i c a n t d i f f e r e n c e s i n
t h e a c t i v i t y g r a d i e n t s of t h e s o i l enzymes i n t h e v e g e t a t i o n t y p e s
under study.
The f o r e s t a n d s h r u b l a n d s o i l s had h i g h e r c l a y c o n t e n t i n t h e
t o p s o i l t h a n t h e i r g r a s s l a n d c o u n t e r p a r t ( T a b l e 41, i n d i c a t i n g
t h a t c l a y p a r t i c l e s were p r o b a b l y more e a s i l y t r a n s l o c a t e d a n d washed
away from t h e g r a s s l a n d s o i l .
Although s o i l p a r t i c l e s are known t o
b i n d s o i l enzymes (McLaren, 1 9 7 5 ) , t h i s a p p a r e n t s e l e c t i v e removal
a
0 .
.4
rn-
*
hC
.G r
a
3
al --a
5
cn
s
0
0 In
rn
Yl
al
I
rl
0
C
rl
1
*a - rn
a
.4
a l o
x rn
alr
0
C
*wX 8
o f c l a y p a r t i c l r s from s u r f a c e g r a s s l a n d s o i 1 d i d n o t seem t o
i n f l u e n c e amylase a n d c e l l u l a s e a c t i v i t i e s t o a n y a p p r e c i a b l e e x t e n t
s i n c e t h e a c t i v i t i e s of t h e s e c a r h o h y d r a s e s w e r e s i g n i f i c a n t l y
h i g h c r i n g r a s s l a n d s o i l t h a n i n .shrubland o r f o r e s t s o i l s ( T a b l e s
5 , 6, 7 and 8 ) .
A g r e a t e r percentage of t h e sand f r a c t i o n i n
g r a s s l a n d compared t o f o r e s t and s h r u b l a n d s o i l w a s c o a r s e .
I n f l u e n c e o f V e g e t a t i o n Cover a n d S o i l O r g a n i c Matter on
Amylase and C e l l u l a s e A c t i v i t i e s *
The a c t i v i t i e s o f amylase and c e l l u l a s e w e r e h i g h e s t i n
g r a s s l a n d s o i l , i n t e r m e d i a t e i n .dhrubland s o i l and l o w e s t i n f o r e s t
s o i l throughout t h e y e a r of study (Tables
5,
6,
7
and 8 ) .
This
t r e n d w a s s i m i l a r t o t h e r e s u l t s o f Nizova (1961) and Ross ( 1 9 6 6 ) who
found a c t i v i t i e s o f enzymes h y d r o l y z i n g s u c r o s e and s t a r c h g r e a t e r
under g r a s s e s t h a n f o r e s t .
These d i f f e r e n c e s have been a t t r i b u t e d
t o d i f f e r e n c e s i n t h e composition o f p l a n t m a t e r i a l s f a l l i n g n a t u r a l l y
on s o i l s u n d e r t h e t h r e e c o n t i g u o u s v e g e t a t i o n c o v e r s .
I n c o n t r a s t t o t h e t r e n d i n enzyme a c t i v i t i e s , o r g a n i c m a t t e r
was
soil
h i g h e s t i n f o r e s t , i n t e r m e d i a t e i n shrubland and lowest i n g r a s s l a n d
able 4 ) .
I t i s known t h a t s o i l p a r t i c l e s t h a t b i n d s o i l
enzymes impose s t e r i c r e s t r i c t i o n s which r e s u l t i n slow d i f f u s i o n
o f s u b s t r a t e s t o t h e i m m o b i l i z e d enzyme s i t e (Ladd a n d B u t l e r ,
1975).
Thus t h e s u b s t r a t e c o n c e n t r a t i o n i n t h e immediate v i c i n i t y o f t h e
a t t a c h e d enzyme i s u s u a l l y low a n d t h e enzyme i s c o n s i d e r e d t o be i n
e x c e s s a t any one t i m e ( ~ c ~ a r eand
n S k u j i n s , 1971).
Consequently,
Table
5.
Mean amylase a c t i v i t y i n s o i l s under d i f f e r e n t
vegetation covers.
Mean amylase a c t i v i t y
(mg g l u c o s e r e l e a s e d / g dry
s o i l p e r 5 h)
V e g e t a t i o n Cover
Table 6.
Forest
0.01077
Shrubs
0.01927
Grasses
0.02836
Monthly amylase a c t i v i t y i n .=oil under d i f f e r e n t
vegetation covers.
Mean amylase a c t i v i t y
Month
Grass1 and
Shrub1and
Forest
mg glucose/g dry s o i l p e r 5 h
87
0.0189
0.0098"~
O.OO5O
%ir.
87
0.0204
0.0 12oaC
0.0065
Apr.
87
0.0209
0.0112~~
b
0. (W50
*Y
87
0.0224
0.0128~~
Jun. 87
0.0345
0.0209~
87
0.0461
0.0371aC
87
Sep. 87
0.0524
0.0~+18~~
0.0407
0.0316~~
O C ~ .87
0 -0245
0.0203~~
Jul.
Aug-
Nov.
87
0.0202
D~C.
87
0.0201
0.0 120aC
ac
O.OlO!j
Jan.
88
O.ol94
0.0112~~
C.V.
a
b
Feb.
(%)
41.9
59
b
0.0079~
b
0.0115
b
0.0208
b
0.0281
0.0148~
b
0.0111
b
0.0057
b
0.0064
b
0.0064
67
- --.
--
-
-
D i f f e r e n c e between g r a s s l a n d and s h r u b l a n d s i g n i f i c a n t a t 0.05
b ~ i f f e r e n c ebetween g r a s s l a n d and f o r e s t s i g n i f i c a n t a t 0.05
C
D i f f e r e n c e between shrubland and f o r e s t s i g n i f i c a n t a t 0.05
level
level
level
--
--
Table
7.
Mean c e l l u l a s e a c t i v i t y i n s o i l s u n d e r d i f f e r e n t
vegetation covers,
- -
-
V e g e t a t i o n Cover
(mg
Mean c e l l u l a s e a c t i v i t y
g l u c o s e . r e l e a s e d / g d r y s o i l p e r 24 h )
.-.
- --
Forest
Shrubs
Grasses
T a b l e 8.
0.06475
Monthly c e l l u l a s e a c t i v i t y i n s o i l u n d e r d i f f e r e n t
vegetation covers.
Mean
Grassland
Yonth
c e l l u l n s c ;\c-t,ivity
&rubland
mg g l u c o s e / y
Feb.
gF
Yar.
87
Apr-
Forest
d r y s o i l p e r 21t h
.
Yay
g3
J u n . $2
Jul.ql
Aug.
ST
Sep. g 1
O c t . 5?+
Nov.
87
oec.
$7
88
Jan.
C.V.
a
D i f f e r e n c e b e t w e e n q r a s s l a n d a n d s h r u l ~ l a n t ls i g n i f i c a n t a t 0.07
b
C
(%)
level
D i f f e r e n c e b e t w e e n g r a s s l a n d a n d f o r e s t s i g n i f i c a n t a t 0.05
level
1)i f f e r e n c e b e t w e e n s h r u b l a n d a n d f o r e s t s i g n i f i c a n t a t 0.05
level.
NS
?ion-significant
difference.
I
.
r
although the organic matter content of the forest soil was higher
than in savanna soils (that is, soils under shrubs and grasses),
the effective concentration of organic substrates at the catalytic
site of the carbohydrases may not have necessarily been higher
for forest than savanna soils.
Also, it is important to note that
organic matter determinations are bulk soil measurements whose
values may differ from those at the molecular- or micro-site of the
inmobilized soil enzymes.
Analysis of the composition of mature plant litter collected
from the canopy of each of the vegetation covers showed that leaf
b
litter from the forest floor had a much lower cellulose to lignin
and starch to lignin ratios than did litter from shrubland and
grassland vegetation covers (Table 9).
Lignin inhibits the multi-
plication of soil microorganisms, particularly fungi (~dachi&
d.,
1987) when it is present in high amounts in plant residues returning
to the soil.
Due to the relatively high lignin content of forest
litter, it is likely that organisms that decompose starch and
cellulose would be more active under savanna vegetation and thus amylase
and ccllulase activities would be expected to be-higher under grasses
and shrubs than under forest vegetation.
Also, in undegraded plant
litter, the close association of lignin with polysaccharide~,
particularly in mature plant tissues, protects the polysaccharides
from hydrolysis by soil polvsaccharidases (~orkrans,1967;
Wardrop, 1971).
Although liynin is resistant to decomposition it is
not however i m n e to it,
Lignin undergoes slow decomposition to give
T a b l e 9.
P e r c e n t a g e s o f d i f f e r e n t o r g a n i c components
i n leaf l i t t e r c o l l e c t e d from t h e f l o o r s o f
different vegetation covers.
Grassland
Shrubland
Forest
Cellulose
25 .0
28.4
36.0
Starch
14.5
13.2
11.6
Lignin
6.0
8. 0
13-5
--4
Ratios
>-
phenolic and quinonoid compounds which combine with amino acids
to form humus (Christmm and Oglesby, 1971; Martin &ELL.,
Lignin and its slow-decomposition products
acids
-
-
1980).
phenolic and humic
are known to bind soil enzymes and decrease their activities
(Ladd and Butler, 1975).
The binding of soil enzymes on such soil
organic materials could be such as to make the active site of the
enzyme not exposed to substrate binding or it could induce conformational changes on the enzyme molecule such that the substrate does
not fit into the enzyme active site.
Although the content of
phenolic compounds in the soils under study were not determined the
relatively lower pH of the forest than grass2and soil (Table
11)
may be an indication of accumulation of phenolic and humic acids in
forest soil arising from liqnin decomposition.
Martin
aJ.,
(1980)
showed that during lignin decomposition, residual lignin carbons
were incorporated into humic acid fractions.
Thus, the relatively
high lignin content of forest litter (Table 9) may thus, at least
partially, explain the relatively low amylase and cellulase activities
in forest soil.
Similar to results obtained by Pancholy and Rice (1973a), the
activities of amylase and cellulase did not correlate positively with
amounts of organic matter
khan (1970).
able
10) as previously reported by
These results are consistent with those of Arnato &
d.,(1987) who observed that differences in t h ~decomposition rates
of different plant materials in soils were related positively with
-78rainfall but showed no such relationship with soil organic carbon.
The highly significant negative correlation between amylase and
cellulase activities on the one hand and the amounts of soil organic
matter within each vegetation type on the other (Table
lo), could
be pertially explained from the point of view that the activities
of the enzymes under study may have been responding to monthly
changes in soil moisture (the trend of which was opposite to that
of organic matter) rather than to changes in soil organic matter
levels within each vegetation cover.
Highly significant and positive
multiple correlations were observed amongst soil moisture,organic
+
matter and amylase or cellulase activity (Table ll), thusindicating
that soil moisture was probably more important in regulating the
enzyme activities under study than organic matter levels.
Organic
matter levels in soils under the three vegetation covers increased
as dry season progressed (Tables 1 , 2 and 3 ) but enzyme activities
were decreased by the soil moisture stress occurring during those
dry months.
During the wet months the activities of amylase and
cellulase enzymes increased significantly (Figs 1 and 2) due to
increases in soil moisture, although the organic matter levels were
relatively lower than during the dry months within each vegetation
cover.
The negative correlation between the carbohydrase activities
and the soil organic matter content could also be due to the response
of the enzymes to changes in the
composition of plant materials
returning to the soil with time.
Crowder (1974) showed that during
-79t h e r a i n y season t h e l e a f m a t e r i a l o f t r o p i c a l p l a n t s contained
h i g h amounts o f c a r b o h y d r a t e s b u t a s d r y s e a s o n p r o g r e s s e d t h e p l a n t s
became p r o g r e s s i v e l y l o w e r i n c a r b o h y d r a t e s a n d h i g h e r i n l i g n i n .
I t w a s l i k e l y t h e r e f o r e that a r e l a t i v e l y h i g h l i g n i n c o n t e n t o f
p l a n t materials r e t u r n e d n a t u r a l l y t o t h e s o i l d u r i n g t h e d r y months
would c a u s e a d e c r e a s e i n a m y l a s e a n d c e l l u l a s e a c t i v i t i e s , a l t h o u g h
t h e amount o f s o i l o r g a n i c matter w a s g e n e r a l l y h i g h e r d u r i n g t h a t
p e r i o d f o r each v e g e t a t i o n cover.
The l a c k o f a p o s i t i v e c o r r e l a t i o n
between t h e enzyme a c t i v i t i e s a n d s o i l o r g a n i c matter l e v e l s w i t h i n
e a c h v e g e t a t i o n t y p e , a n d t h e f q c t t h a t t h e f o r e s t s o i l which had
t h e h i g h e s t o r g a n i c m a t t e r l e v e l h a d t h e l o w e s t enzyme a c t i v i t i e s
compared w i t h s o i l s u n d e r s h r u b s a n d g r a s s e s , i n d i c a t e t h a t t h e
a c t i v i t i e s o f a m y l a s e a n d c e l l u l a n e enzymes a p p a r e n t l y w e r e n o t
d e t e n n i n e d i n t h e main by t h e amount o f s o i l o r g a n i c m a t t e r p r e s e n t
a n d t e n d e d t o s u p p o r t t h e s u g g e s t i o n by Ross (1966) a n d P a n c h o l y
and Rice (1973a), t h a t t h e t y p e o f v e g e t a t i o n and t h u s t h e t y p e of
o r g a n i c m a t t e r t h a t e n t e r s t h e s o i l are t h e c h i e f d e t e r m i n a n t s o f
t h e a c t i v i t y g r a d i e n t s o f carbohydrases i n s o i l .
I n f l u e n c e o f S e a s o--n
o i-s.-t- u-r-eon
--- a-n-d -S.o.--i l M
-- -- Amylase a n d C e l l u l a s e
Activities.
The a c t i v i t j es o f thr* r a r b o h v d r a s c s u n d e r s t u d y sl~owcda ~ e a s o n n l
v a r i a t i o n i n s o i l s under t h e t h r e e v e g e t a t i o n t y p e s ( F i g s
A s i ndj
1 and 2 ) .
c4atcd e a r 1 i e r , durincl t h c r n i n v s e a s o n , when ~ o 1 i.; r-ernnincd
m o i s t , a m v l a s e a n d c o l l u l a s c a c t i v i t i e s increased, b u t a t t h e end o f
-80-
the rainy season as soils dried out their activities wcrr again
reduced.
These consistent gradients in activities of the enzymes
under study represent a widespread phenomenon
-
that enzymatic
hydrolysis of soil polysaccharides under field conditions is limited
by dry-season moisture stress.
This would indicate also that the
expression of the soil's biochemical potential could be severely
restricted by unfavourable climatic conditions.
Such seasonal
variation of a soil hydrolase (arylsul~hatase)activity was also
observed in Northern Nigerian soils by Cooper (1972).
These results
are consistent with those of Agbim (1987), who showed that dry&
season field decomposition rates of leaf litter from some West
African plant species (Acioa, Imperata and Pentaclethra) were less
than half those obtained in the moist laboratory condition.
The
highly significant positive correlation between soil moisture as
well a5 total monthly rainfall, and the activities of amylase or
cellulase enzyme (Table 10) further supports the role of the moisture
factor as a major determinant of the level of carbohydrase activities
in soil.
Nevertheless, Ross (1966) observed a highly significant
negative correlation of mean annual rainfall and sucrose-hydrolysing
activities and suggested that the greatest proportions of these
enzymes occur in the organic matter of the drier soils.
He found
no apparent influence of rainfall on starch-hydrolysing enzymes.
b s t enzymatic reactions take place in solution and an adequate soil
moisture is required to provide the medium for the catalysis.
It is
logical therefore that rainfall with the subsequent adequate soil
Months
Fig.1:Seasonal variation of amylase activity in forest -shrub
grass lund mosaic soils(= soil under grasses,
-=
soil under shrubs,
= soil under forest ) and
total monthly ruinfall recorded from February 1987 to
January 1988 (shaded blocks).
-
-
Months
Fig.2:Seasonal variatim of cellulase a c t i i in forest-shrubgrassland m i c soi Is (x-+ =soil under grass- ,
-=soil
under s h r l r b s w = sol1 under forest ) and
total rainfall recorded from February 1987 to
January 1988 (shaded blocks).
-83moisture would b e e x p e c t e d t o i n c r e a s e t h e a c t i v i t i e s o f s o i l
a m y l a s e a n d c e l l u l a s e enzymes.
K i s s e t aL.,
(1978) a l s o concluded
t h a t t h e rate o f b i o c h e m i c a l c o n v e r s i o n s o c c u r r i n g i n a s o i l i s
d e t e r m i n e d l a r g e l y by i t s w e t n e s s .
I t may b e d i f f i c u l t however,
t o a s c e r t a i n t h e c r i t i c a l s o i l m o i s t u r e r e g i m e r e q u i r e d f o r optimum
polysaccharidase a c t i v i t y since these e x t r a c e l l u l a r (abiontic) s o i l
enzymes h a v e been s h o r n ( ~ c ~ a r e n1 9, 7 5 ) t o be s t r o n g l y a d s o r b e d
o n o r g a n i c a n d i n o r g a n i c s o i l p a r t i c l e s which p r o v i d e t h e m o l e c u l a r
e n v i r o n m e n t f o r s o i l enzyme c a t a l y s i s .
The s o i l p a r t i c l e s u r f a c e s
on which t h e s o i l enzymes are i m m o b i l i z e d m a i n t a i n a t h i n w a t e r
b
f i l m a r o u n d them t h a t
may e n a b l e enzyme c a t a l y s i s t o t a k e p l a c e
e v e n when t h e m o i s t u r e s t a t u s o f t h e b u l k s o i l s o l u t i o n may n o t
permit t h e s u r v i v a l o f p l a n t r o o t s and microorganisms d u r i n g t h e
d r y season.
Adequate s o i l m o i s t u r e a r i s i n g from r a i n w a t e r may s t i m u l a t e
p l a n t r o o t development a n d m i c r o b i a l a c t i v i t y l e a d i n g t o a n i n c r e a s e d
s e c r e t i o n o f e x t r a c e l l u l a r enzymes.
I t i s p o s s i b l e however t h a t t h e
r e l a t i v e l y h i g h a c t i v i t y o f a m y l a s e a n d c e l l u l a s e enzymes d u r i n g t h e
w e t months may n o t b e d u e t o
de -n
s y n t h e s i s o f t h e enzymes a l o n e ,
b u t a l s o l a r g e l y as a r e s u l t o f a c t i v a t i o n o f p r e - e x i s t i n g
but
q u i e s c e n t enzymes by s o i l m o i s t u r e a n d s o l u b i l i z a t i o n o f t h e s u b strates w i t h i n t h e s o i l environment.
T h i s is because p o l y s a c c h a r i d a s e s
are t r u e e x t r a c e l l u l a r enzymes ( K i s s e
t
e.,1 9 7 8 ) a n d
the activity
measured a t a n y t i m e r e p r e s e n t s l a r g e l y t h e a c t i v i t y o f a c c u m u l a t e d
-84enzyme l e v e l s i n t h e s o i l ( S k u j i n s ,
1967
) rather than a c t i v i t y
due t o new p r o t e i n s y n t h e s i s by p l a n t r o o t s , s o i l f a u n a a n d m i c r o organisms.
Pancholy and Rice (1973b) demonstrated t h a t a l t h o u g h
t h e t r e n d s w e r e g e n e r a l l y s i m i l a r , t h e a c t i v i t i e s o f amylase,
c e l l u l a s e a n d o t h e r h y d r o l a s e s d i d n o t peak a t t h e same t i m e w i t h
m i c r o b i a l numbers.
Also, i t is p o s s i b l e t h a t previous p l a n t s
c o n t r i b u t e d t o t h e c u r r e n t amylase and c e l l u l a s e c o n t e n t o f t h e
s o i l s , p a r t i c u l a r l y i n s a v a n n a areas which u n d e r g o f r e q u e n t b u r n i n g .
I t i s known t h a t w i t h i n t h e s o i l e n v i r o n m e n t a b i o n t i c enzymes are
s t a b i l i z e d by t h e i r a ~ s o c i a t i o n ~ w i tbho t h o r g a n i c a n d i n o r g a n i c
s o i l c o l l o i d s a n d may p e r s i s t i n t h e s o i l f o r a v e r y l o n g t i m e
( S k u j i n s a n d McLaren, 1968; McLnren,
1975).
I n t h e d r y s e a s o n when
t h e r e is s o i l m o i s t u r e a t r r s s , t h e a c t i v i t i e s o f t h c accumulated
p o l v s a c c h a r i d a s e s would b e low due t o low s o l u b i l i t y o f t h e s u b s t r a t e s .
A s w e t s e a s o n a p p e a r s , t h e s e enzymes c o u l d bccomc a c t i v a t e d a n d t h e
m o i s t e n e d s u b s t r a t e s may b e e a s i l y h y d r o l v s e d due t o a weakening
o f t h e bonds h o l d i n g t h e s u b s t r a t e s t o g e t h e r .
The mechanism o f
a c t i v a t i o n o f s o i l enzymes by s o i l m o i s t u r e i s however n o t unclerstootl.
I t may be d u e t o removal o f p r o t e c t i v e g r o u p s from t h e a c t i v e s i t e
o f q u i e s c e n t s o i l enzymes i n t h e p r e s e n c e o f s o i l w a t e r , t h u s
e.xposing t h e enzyme t o i t s s u b s t r a t e .
It is also possible that
adequate s o i l m o i s t u r e l e a d s t o i n c r e a s e d a c t i v i t y o f s o i l organisms
which e x c a v a t e a n d c h u r n up t h e s o i l .
T h i s was e v i d e n t i n t h e
i n c r e a s e d i n c i d e n c e o f earthworm c a s t s on t h e s o i l s u r f a c e o f a l l t h e
-85t h r e e v e g e t a t i o n t y p e s d u r i n g t h e wct months.
T h i s mixing o f o r g a n i c
a n d i n o r g a n i c s o i l components c o u l d e x p o s c a l i t t l e more crumb m a t r i x
enzyme t o i t s s u b s t r a t e a n d h e n c e i n c r e a s e enzyme s i t e a v a i l a b i l i t y t o
substrate.
Ross a n d C a i r i n s ( 1 9 8 2 ) showed t h a t e a r t h w o r m s s t i m u l a t e
b i o c h e m i c a l a c t i v i t y i n s o i l s which r e s u l t i n i n c r e a s e i n t h e
a c t i v i t y o f some s o i l enzymes i n c l u d i n g a m y l a s e a n d c e l l u l a s e .
S t i m u l a t i o n of p r e - e x i s t i n g b u t q u i e s c e n t amylases h a s been r e p o r t e d
i n u n g e r m i n a t e d m i l l e t (Opoku
&
a.,1983;
Nwankwo, 198ft) a n d
b a r l e y g r a i n s ( B r i g g s , 1 9 6 3 ) on a b s o r p t i o n o f w a t e r by t h e d r y s e e d s
a t t h e beginning of g e m i n a t i o n .
b
The h i g h e s t a m y l a s e a n d c e l l u l a s e a c t i v i t i e s o c c u r r e d b e t w e e n
?lay a n d September w i t h a p e a k i n August ( ~ i ~1 as n d 21, c o r r e s p o n d i n g
t o peak s o i l m o i s t u r e l e v e l s i n t h e t h r e e v e g e t a t i o n t y p e s .
Ccllulasc
a c t i v i t y i n f o r e s t s o i l however peaked i n J u l y , a l t h o u g h t h e r e w a s
no
s i g n i f i c a n t d i f f e r e n c e i n a c t i v i t y between J u l y a n d August.
Honthly v a r i a t i o n i n amylase a c t i v i t y based on c o e f f i c i e n t o f
variation (C.V.),
w a s higher i n f o r e s t s o i l t h a n i n shrubland and
g r a s s l a n d s o i l s ( T a b l e 61, w h i l e s i m i l a r v a r i a t i o n s i n c e l l u l a s e
a c t i v i t i e s rere s l i g h t l y h i g h e r i n g r a s s l a n d s o i l t h a n i n s h r u b l a n d
o r f o r e s t s o i l (Table 8 ) .
r e a d i l v apparent.
The r e a s o n s f o r t h e s e d i f f e r e n c e s are n o t
Ross ( 1 3 6 6 ) s u g g e s t e d t h a t v a r i a t i o n s i n s o i l
m o i s t u r e c o n t e n t o v e r a l o n g p e r i o d c a n h a v e marked e f f e c t s on s o i l
enzyme a c t i v i t y .
was lhh.lt5
cm.
The a n n u a l p r e c i p i t a t i o n i n Nsukka d u r i n g 1987
From F e b r u a r y 1987 t o J a n u a r y 1988 r a i n f e l l f o r 108
-86day7 o u t o f which t h e r e w e r e o n l y 1 2 day? o f r a i n t h r o u g h o u t t h e
months o f Feb, Mar-,
S i n c e Nsukka s o i l s are
Apr, Nov, Dec, a n d Jan.
sandy, porous and w e l l d r a i n e d , t h e l e n g t h o f t h e r a i n y season and
h e n c e t h e l e n g t h o f t i m e t h e s o i l r e m a i n s wet may b e more i m p o r t a n t
i n determining t h e a c t i v i t y g r a d i e n t s o f amylase and c e l l u l a s e
enzymes i n t h e s e s o i l s , t h a n t h e a n n u a l r a i n f a l l .
D u r i n g t h e months o f March a n d May t h e r e w e r e w i d e l y d i s p e r s e d
r a i n s t o r m s o c c u r r i n g on
7 Mar, 2 1 Mar, 2 9 M a r ,
1 May,
8 May, 17
Hay a n d 27 May w i t h c o r r e s p o n d i n g v a l u e s o f 0.8 c m , 0.36
0.1 cm,
1.3 c m , 0.43 c m a n d 0.94 cm.
cm, 0.9 c m ,
S o i l s w e t t e d by s m a l l a n d
b
i n f r e q u e n t r a i n s would d r y o u t w i t h i n a r e l a t i v e l y s h o r t p e r i o d .
Such a l t e r n a t e w e t a n d d r v c o n d i t i o n s a r e known t o g i v e rise t o
enhanced m i n e r a l i z a t i o n o f s o i l o r g a n i c m a t t e r ( l l i r c h , 1958).
p r o b a b l y e x p l a i n s t h e g e n e r a l sudden
This
rise i n a c t i v i t i e s o f amylase
a n d c e l l u l a s e enzymes i n March a n d a g a i n i n May i n s o i l s u n d e r t h e
three v e g e t a t i o n t y p e s ( ~ i g s 1 a n d 2 ) .
Cooper ( 1 9 7 2 ) a l s o e x p l a i n e d
t h e rise i n a r y l s u l p h a t a s e a c t i v i t y between
5
Febeand 4 May i n
Northern Nigerian s o i l s t o a l t e r n a t e drying and s o i l wetting.
However,
l i t t l e i s known a b o u t t h e mechanism i n v o l v e d .
R e l a t i o n s h i p s o f Amylase and C e l l u l a s e A c t i v i t i e s t o
Other S o i l Factors.
T o t a l n i t r o g e n , a v a i l a b l e phosphorus, c a t i o n exchange c a p a c i t y
(CEC) a n d t o t a l e x c h a n g e a b l e b a s e s (TED) o f t h e s o i l s u n d e r e a c h o f
t h e t h r e e v e g e t a t i o n c o v e r s showed t h e same t r e n d a s o r g a n i c m a t t e r .
That is, the values were highest in forest soil, and lowest in
grassland soil.
This would be expected since in humid tropical
soils, organic matter is the major contributor
chemical properties.
to such soil
Soil pH was however lowest in forest soil and
highest in grassland soil (Table 4 ) reflecting probably, the higher
liynin content and hence phenolic and humic acids of the forest
litter.
Tt has earlier been shown that organic matter level had a
significant negative correlation with enzyme activity(lab1e 10). As with
organic matter, most other soil factors for which organic matter
6
level in humid tropical soils plays a major role in their expression
also showed negative correlations with the carbohydrase activities
(Table 10).
Significant hut negative correlations wcrr obtained
between amylase and c c l l u l a s e activities on the one hand and each
of thc following soil factors: pH, C/N ratio, per cent base saturation ('% BS) and, exc.ept for forest soil, total exchangeable bases
(TED).
High C/N ratios generally decrease the activity of micro-
organisms on organic matter decomposition although fungi which are
the main decomposers o r plant materials in the humid tropics are
relatively adapted to high C/N ratios of organic materials (~lexander,
1977).
However, how C/N ratios of organic materials may affect the
abilitv of soil microorganisms to svnthesiz~and secrete cxtracellular
enzymes is not understood.
Studies by Agbirn (personal communications) and Agbim (1987)
indicate that significant increases in pH could be obtained by the
-88a d d i t i o n o f o r g a n i c matter t o p o o r l y b u f f c r e d s o i l s a s ar-e f o u n d
i n NsuMca.
T h e d e p e n d e n c e of p H o n s o i l o r g a n i c m a t t e r may t h u s
t o some e x t e n t e x p l a i n t h e n e g a t i v e c o r r e l a t i o n s o b s e r v e d b e t w e e n
t h e s o i l enzyme a c t i v i t i e s ancl s o i l pll ( ~ a b l c10). Thc r e s u l t s r e p o r t e d b y
Ross (1975), i n d i c a t e t h a t t h e r e l a t i o n s h i p ( p o s i t i v e o r n e g a t i v e )
b e t w e e n s o i l pH a n d c a r b o h y d r a s e a c t i v i t i e s d e p e n d e d o n t h e s o i l
twe
I n h u m i d t r o p i c a l s o i l s i n w h i c h low a c t i v i t y c l a y s p r e d o m i n a t e ,
o r g a n i c matter c o n t r i b u t e s m o s t o f t h e b a s e s a t u r a t i o n a n d TEB.
S i n c e o r g a n i c matter w a s n e g a t i v s l y c o r r e l a t e d w i t h a m y l a s e a n d
c e l l u l a s e a c t i v i t i e s , i t would be l o g i c a l t h e r e f o r e t o a l s o e x p e c t
a n e g a t i v e c o r r e l a t i o n w i t h % H S o r TER.
c o r r e l a t i o n b e t w e e n TER o r
57
However, t h e
negative
I3S a n d t h e c a r b o h y d r a s e a c t i v i t i e s
( T a b l e 1 0 ) may h a v e a l s o r e s u l t e d f r o m t h e l e a c h i n g l o s s e s o f b a s e s
a t h i g h m o i s t u r e l e v e l s c o r r e s p o n d i n g t o i n c r e a s e i n t h e enzyme
activities.
T h e enzyme a c t i v i t i e s w e r e h i g h e s t i n g r a s s l a n d s o i l
( w h i c h a l s o had t h e l o w e s t o r g a n i c matter l e v e l , t h e l o w e s t TEB
b u t t h e h i g h e s t % BS) a n d l o w e s t i n f o r e s t s o i l w h i c h h a d t h e h i g h e s t
o r g a n i c m a t t e r l e v e l a n d TEB ( F i g s
1 and 2, Table
4).
Within each
v e g e t a t i o n t y p e however, t h e s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n s
o b t a i n e d b e t w e e n t h e enzyme a c t i v i t i e s a n d % BS o r TER o r b o t h are
i n d i c a t i v e o f some o f t h e d i f f i c u l t i e s t h a t may b e e n c o u n t e r e d i n
u s i n g t h e a c t i v i t i e s o f t h e s e enzymes t o p r e d i c t n u t r i e n t s t a t u s o f
the soils.
By t h e m s e l v e s , p a r t i c u l a r enzyme l e v e l s d o n o t n e c e s s a r i l y
s i g n i f y p a r t i c u l a r l e v e l s o f s o i l f e r t i l i t y (Fjurns,
19781, b u t r a t h e r
-90biochemical potential.
The expression of this potential can be
governed by many factors, including climatic properties.
Skujins
(1978) also pointed out that enzymes are substrate-specific and
individual enzyme measurements cannot reflect thc total nutrient
status of the soil: individual soil enzyme measurements would
however answer questions regarding specific decomposition processes
in the soil or questions about specific nutrient cycles.
suggested (Kiss
et &.,
It was
1978) that carbohydrases would indicate
decomposition rates of litter and hence rate of mineralization of
organic matter.
Cation exhange capacity (CEC) values were positively, although
in most cases not significantly, correlated with the carbohydrase
activities (Tahle 1 0 ) .
This is in contrast with the situation
' US and TED.
ohtained with %
This would suqgcst that under the
conditions of this study, CEC values were less dependent on soil
organic matter than %
' D S and TED.
I
This inclecd appears to be the case
13 of the soils'
since under the three vegetation types, 69.2 to 83.'J%(~able
exchange sites were occupiecl by acid-forming cations the sources
l! ions).
of which would be mainly inorganic ( ~ and
1
It is not clear why available phosphorus was positively
correlated with amylase and cellulasr activities
able
20) if, as
ha= heen suggested (.Sanchez, 29761, a largcr fraction of the phosphorus
in tropical soils is associated with soil organic matter.
In
plant residues, organic phosphate compounds are usually associated
with carbohydrates and are enclosed within a cellulose cell wall.
These
ynt~
, fructose phocyhate, actone
c o ~ p o u n d sinclude p l , ~ ~ ~ [: h; o~~ h
r!!os~i:ate, AT1 e tc.
A priming action of cellulase and amylase enzymes on plant residues
would probably have exposed the organic phosphates to the action of
soil phosphatases with the concomitant release of available phosphate
from organic sources.
This appears to account, at least in part,
for the positive and significant correlation observed between the
enzyme activities and available P in the three vegetation types
(Table 10).
Ross (1975) showed a significant positive relationship
between glycoside hydrolase and phosphatase activities in soils.
Multiple correlation coefficients (Table 11) were generally
stronger than simple correlation coefficients (Table 10) between the
b
soil carbohydrase activities and soil moisture or organic matter in
the three-vegetationtypes, meaning that the three factors
-
soil
moisture, organic matter and carbohydrase activities were more
closely related together than organic matter or soil moisture
factor acting singly on amylase or cellulase activity.
It is
apparent therefore that the combined influence of soil moisture and
organic matter were more important in determining the activity
gradients of amylase and cellulase activities in the soils under
study than each of these two soil factors acting singly on the enzyme
activities.
Nevertheless, the significant correlations recorded
in Table 10 tend to indicate that many factors may be responsible
for the levels of amylase and cellulase activities in the soils under
study.
Any relationship of amylase and cellulase activities to the
other soil chemical properties may however be dependent on the general
Table 11.
Multiple correlation coefficients ( R ) for
carbohydrase activities, soil moisture and
organic matter levels in the three vegetation
types.
I
Multiple correlation Coefficient ( R )
Cel lulase
L
0.99**
Forest
Shrubland
Grassland
1
0.96* *
0.98* *
0.84**
0.92"'
Significance of correlation coefficients for n = 12
*.Significant
at 1% probability level
-
biochemical metabolism i n t h e s o i l under a p a r t i c u l a r v ~ ~ c t a t i o n
c o v e r , a n d p r o b a b l y on g e a s o n a l o r s h o r t - t e r m e n v i r o m c n t a l e f f e c t s
as w e l l .
V a l u e s o f s o i l a m v l a s ~a n d c e l l u l a s e a c t i v i t i e s r e p o r t e d by
s e v e r a l w o r k e r s a r e q u i t e v a r i a b l e (ROSS, 1965; 1 9 6 6 ; 1 9 7 6 ;
fhwfield,
1971; Pancholy and Rice,
and Miyashita,
1973a; Hayano, 1986; Kanazawa
1 9 8 7 ) , d e p e n d i n g o n t h e a s s a y method a d o p t e d a n d
d i f f e r e n c e s a r i s i n g from s o i l h e t e r o g e n e i t y .
Ross ( 1 9 6 8 ) , s u g g e s t e d
t h e p o s s i b l e r o l e o f glucose oxidase i n i n t e r f e r r i n g i n a s s a y s of
o t h e r s o i l enzymes i n w h i c h t h e ~ r o d u c t i o no f g l u c o s e was measured.
Most p o l y s a c c h a r i d a s e s h y d r o l y s e t h e i r s u b s t r a t e s e v e n t u a l l y t o
glucose.
I t i s p o s s i b l e t h a t some o f t h e g l u c o s e r e l e a s e d may b e
f u r t h e r metabolized t o gluconic a c i d through t h e a c t i v i t y of n a t u r a l l y
o c c u r r i n g g l u c o s e o x i d a s e i n t h e s o i l , t h u s r e d u c i n g t h e measured
carbohydrase a c t i v i t y .
S i n c e many o f t h e a s s a y s o f c a r b o h y d r a s e
enzymes depended upon t h e measurement o f r e d u c i n g s u g a r s , t h e
s t a b i l i t y o f t h e s u g a r s p r o d u c e d would b e e s s e n t i a l f o r a c c u r a t e
results.
A l t h o u g h n o a t t e m p t w a s made i n t h i s s t u d y t o estimate t h e
a c t i v i t y o f n a t u r a l l y occurring glucose oxidase i n t h e s o i l s under
s t u d y , i t is l i k e l y t h a t i t s e f f e c t on t h e measured amylase and
cellulase a c t i v i t i e s were negligible.
T h i s i s because i n amylase
a s s a y s l a r g e a m o u n t s o f g l u c o s e are u s u a l l y p r o d u c e d (ROSS a n d
Roberts,
1968; 1970) a n d a n y l o s s o f g l u c o s e t h r o u g h t h e a c t i v i t y
o f g l u c o s e o x i d a s e would l i k e l y b e n e g l i g i b l e .
Also, a l t h o u g h t h e
p r o d u c t i o n o f g l u c o s e i s slow i n c e l l u l a s e a s s a y s (Ross, 1974),
-94glucose oxiciase is not likely to affect quantitative assessment of
cellulase activity in the soils under study since the incubation
period (24 hours) was relatively short.
Ross
(19741, stated that
0
an incubation period of 48 hours at 3 0 C was satisfactory for assaying
soil cellulase activity in the presence of glucose oxidase.
-95-
CONCLUSIONS
From this study it is apparent that the decomposition by soil
polysaccharidases of plant residues falling naturally on the soil
surface depended chiefly on the type of plant material and the time
of year of its fall unto the soil, while the amount involved may
play a less significant role.
The work also suggests that the
activity gradients of the soil polysaccharidases (amylase and
cellulase) are regulated mainly by rainfall and hence soil wetness.
The use of crop residues to improve organic matter levels in humid
tropical soils is thought to likely have a wider application than
b
the use of farm-yard manure.
However, due to the relatively low
amylase and cellulase activities in soils during the dry season,
it is likely that application of high amounts of mature or lignified
plant materials to soil either as mulches or organic amendments
during this period could lead to undesirable accumulation of the
material on the soil.
The large quantities of microorganisms that
may be attracted by the accumulated plant material could cause a
net immobilization of soil nutrients to the detriment of growing
crops.
Placement of large quantities of resistant mature plant
residues near crops in the dry season is therefore not likely to
be a judicious practice.
Placement of herbaceous plant residues
like legumes on the soil surface during the dry season would, as
with resistant plant materials, conserve soil moisture and reduce
soil temperatures.
However, the latter group would have the dis-
advantage of causing some nutrient immobi lization nt least on the
short-term.
I n t h e r a i n y season w i t h r e l a t i v e l y h i g h amylase and
cellulase activities,
i t i s a p p a r e n t . t h a t lcss l i m i t a t i o n would b e
p l a c e d on t h e amount a n d t y p e o f o r g a n i c m a t t e r t h a t o n e c o u l d a d d
t o t h e s o i l , as long a s i t i s economically f e a s i b l e t o t h e farmer
a n d t h e material i s n o t known t o b e t o x i c t o c r o p p l a n t s upon
decomposition.
During t h e r a i n y season, h i g h carbohydrase a c t i v i t y
would l i k e l y l e a d t o a r a p i d m i n e r a l i z a t i o n o f h e r b a c e o u s p l a n t
m a t e r i a l s and hence a ready supply o f carbon and energy t o microorganisms and n u t r i e n t s t o p l a n t s .
L i g n i f i e d p l a n t materials would
o n t h e o t h e r h a n d , b e s l o w l y c o n v e r t e d t o humus d u r i n g t h e w e t
b
scason and have a d e s i r a b l e e f f e c t t o crop p l a n t s .
P l a n t s may i n f l u e n c e t h e enzyme a c t i v i t i e s o f s o i l d i r e c t l y b y
s u p p l y i n g enzymes t h r o u g h t h e i r r o o t e x u d a t e s .
F u r t h e r work i s
n e c e s s a r y t o assess t h e r e l a t i v e a m o u n t s o f enzymes c o n t r i b u t e d
t o t h e s o i l by growing c r o p p l a n t s w i t h i n t h e f o r e s t - s a v a n n a e c o t o n e
a n d t o e s t a b l i s h how t h e t . r a d i t i o n a l s o i l management p r a c t i c e s
i n f l u e n c e t h e a c t i v i t i e s o f t h e s e enzymes i n t h e soi1.s.
Such s t u d i e s
could a s s i s t considerably i n i n t e r p r e t i n g t h e significance of s o i l
enzyme d a t a .
I t would b e n e c e s s a r y a l s o t o p e r f o r m r o u t i n e s o i l
enzyme m e a s u r e m e n t s a n d t h e n c o r r e l a t e enzyme l e v e l s t o t h e d e g r e e
o f o r g a n i c matter m i n e r a l i z a t i o n .
T h i s c o u l d e n a b l e u s t o make
a l l o w a n c e f o r p l a n t n u t r i e n t s t h a t would b e r e l e a s e d from o r g a n i c
s o u r c e s o v e r a g r o w i n g s e a s o n when making f e r t i l i z e r recommendations.
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