Rhizopus Soft Rot on Pear (Pyrus serotina) Caused by Rhizopus stolonifer in Korea


Kwon, J-Hyeuk.; Lee, C-Jung.

Mycobiology 34(3): 151-153

2006


Rhizopus soft rot caused by Rhizopus stolonifer occurred on pears (Pyrus serotina) in the Jinju City Agricultural Products Wholesale Market in Korea from 2004 to 2005. The infection usually started from wounds due to cracking at harvest time. The lesions started as water-soaked, rapidly softened, then gradually expanded. The mycelia grew vigorously on the surface of the fruits and formed stolons. Colonies on potato dextrose agar at 25℃ were white cottony to brownish black. Sporangia were globose, black and 90~120 µm in size. Sporangiophores were light brown and 480~2600 × 12~18 µm in size. Sporangiospores were globose to oval, brownish, streaked, and 8~14 × 6~10 µm in size. Columella were light brownish gray, hemispherical and 70~80 µm in size. On the basis of these symptoms, mycological characteristics and pathogenicity tests on host plants, the fungus was identified as Rhizopus stolonifer (Ehrenb.) Vuill. This is the first report of rhizopus soft rot on pear (P. serotina) caused by R. stolonifer in Korea.

Mycobiology
34(4):
159-165
(2006)
Copyright
©
2006
by
The
Korean
Society
of
Mycology
Bioconversion
of
Lignocellulose
Materials
C.
Pothiraj*,
P.
Kanmani
and
P.
Balaji
l
Dept.
of
Microbiology,
VHNSN
College
626001,
Tamilnadu,
S.
India
`Research
Center
in
Botany,
Thiagarajar
College
(Autonomous),
Madurai
-
625
009,
S.
India
(Received
September
6,
2006)
One
of
the
most
economically
viable
processes
for
the
bioconversion
of
many
lignocellulosic
waste
is
represented
by
white
rot
fungi.
Phanerochaete
chtysosporium
is
one
of
the
important
commercially
cultivated
fungi
which
exhibit
varying
abilities
to
utilize
different
lignocellulosic
as
growth
substrate.
Examination
of
the
lignocellulolytic
enzyme
profiles
of
the
two
organ-
isms
Phanerochaete
chtysosporium
and
Rhizopus
stolonifer
show
this
diversity
to
be
reflected
in
qualitative
variation
in
the
major
enzymatic
determinants
(ie
cellulase,
xylanase,
ligninase
and
etc)
required
for
substrate
bioconversion.
For
example
P
chtysosporium
which
is
cultivated
on
highly
lignified
substrates
such
as
wood
(or)
sawdust,
produces
two
extracellular
enzymes
which
have
associated
with
lignin
deploymerization.
(Mn
peroxidase
and
lignin
peroxidase).
Conversely
Rhizopus
stolonifer
which
prefers
high
cellulose
and
low
lignin
containg
substrates
produce
a
family
of
cellulolytic
enzymes
including
at
least
cellobiohydrolases
and
f
l-glucosidases,
but
very
low
level
of
recognized
lignin
degrading
enzymes.
KEYWORDS:
Bioconversion,
Bio-fuel,
Cellobiohydrolases,
Lignocellulosic
enzymes,
White
rot
fungi
Lignocellulose
Lignocellulose
is
the
major
structural
component
of
woody
plants
and
non-woody
plants
such
as
grass
and
represents
a
major
source
of
renewable
organic
matter.
Lignocellu-
lose
consists
of
lignin,
hemicellulose
and
cellulose.
The
chemical
properties
of
the
components
of
lignocellulosics
make
them
a
substrate
of
enormous
biotechnological
value
(Malherbe
and
Cloete,
2003).
Large
amounts
of
lignocel-
lulosic
"waste"
are
generated
through
forestry
and
agricul-
tural
practices,
paper-pulp
industries,
timber
industries,
and
many
agro
industries.
But,
they
pose
an
environmen-
tal
pollution
problem.
Sadly,
much
of
the
lignocellulose
waste
is
often
disposed
by
biomass
burning,
which
is
not
restricted
to
developing
countries
alone,
but
is
considered
a
global
phenomenon
(Levine,
1996).
However,
the
huge
amounts
of
residual
plant
biomass
considered
as
"waste"
can
potentially
be
converted
into
various
different
value
added
products
including
bio
fuels,
chemicals,
cheap
energy
sources
for
fermentation,
improved
animal
feeds
and
human
nutrients.
Lignocellulytic
enzymes
also
have
significant
potential
applications
in
various
industries
including
chemi-
cals,
fuel,
food,
brewery
and
wine,
animal
feed,
textile,
laundry,
pulp
and
paper
and
agriculture.
This
review's
main
focus
is
to
highlight
significant
aspects
of
lignocellulolytic
biotechnology
with
emphasis
on
demonstrating
the
potential
value
from
an
application
rather
than
basic
research
perspective.
Aspects
which
will
be
reviewed
in
this
article
include:
an
overview
of
some
of
the
major
potential
lignocellullose
derived
high
value
*Corresponding
author
<E-mail:
>
bioproducts;
solid
state
fermentation
processing
as
a
rele-
vant
for
developing
countries;
some
back-ground
on
ligno-
cellulolytic
organisms
and
their
enzymes,
and
finally
looking
at
cost
of
enzymes
and
potential
of
modern
approaches
which
could
be
employed
to
reduce
cost.
Potential
bioproducts
and
their
applications:
Biomass
can
be
considered
as
the
mass
of
organic
material
from
any
biological
material
by
extension
and
any
large
mass
of
biological
matter.
A
wide
variety
of
biomass
resources
are
available
on
our
planet
for
conversion
into
bioprod-
ucts.
These
may
include
whole
plants,
plant
parts
(eg.
seeds,
stalks),
plant
constituents
(eg.
starch,
lipids,
protein
and
fibre),
processing
byproducts
(distiller's
grains,
corn
solubles),
materials
of
marine
origin
and
animal
byprod-
ucts,
municipal
and
industrial
wastes
(Smith
et
al.,
1987).
These
resources
can
be
used
to
create
new
biomaterials
and
this
will
be
required
an
intimate
understanding
of
the
composition
of
the
raw
material
whether
it
is
whole
plant
or
constituents,
so
that
the
desired
functional
elements
can
be
obtained
for
bioproduct
production.
There
are
some
excellent
and
comprehensive
literature
(Bhat,
2000;
Sun
and
Cheng,
2002;
Wong
and
Saddler,
1992a,
b;
Beauchemin
et
al.,
2001, 2003;
Subramaniyan
and
Prema,
2002;
Beg
et
al.,
2001)
available
on
the
differ-
ent
potential
bioproducts
and
their
many
applications but
only
a
few
of
the
high-value
products
will
be
reviewed.
Chemicals:
Bioconversion
of
lignocellulosic
wastes
could
make
a
significant
contribution
to
the
production
of
organic
chemicals.
Over
75%
of
organic
chemicals
are
produced
from
five
primary
base-chemicals:
ethylene,
propylene,
159
160
Pothiraj
et
al.
benzene,
toluene
and
xylene
which
are
used
to
synthesis
of
other
organic
compounds,
which
in
turn
are
used
to
produce
various
chemical
products
including
polymers
and
resins
(Coombs,
1987).
The
aromatic
compounds
might
be
produced
from
lignin
whereas
the
low
molecular
mass
aliphatic
compounds
can
be
derived
from
ethanol
pro-
duced
by
fermentation
of
sugar
generated
from
the
cellu-
lose
and
hemicellulose.
Based
upon
the
predicted
catabolic
pathway
and
the
known
metabolism
of
of
lignin
by
Phanerochaete
chry-
sosporium.
Ribbons
(1987)
presented
a
detailed
discus-
sion
of
the
potential
value
added
products
which
could
be
derived
from
lignin.
Vanillin
and
gallic
acid
are
the
two
most
frequently
discussed
monomeric
potential
products
which
have
attracted
interest.
Vanillin
extraction
from
Vanilla
pods
costs
between
$1200
to
$4000
per
kilo
gram
whereas
synthetic
vanillin
costs
less
than
$15
per
kilo-
gram
(Walton
et
al.,
2003).
Vanillin
is
used
for
various
purposes
including
being
an
intermediate
in
the
chemical
and
pharmaceutical
industries
for
the
production
of
herbi-
cides,
anti-foaming
agents
or
drugs
such
as
papaverine,
L-
dopa
and
the
anti
microbial
agent,
trimethoprim.
It
is
also
used
in
household
products
such
as
air-fresheners
and
floor
polishes
(Walton
et
al.,
2003).
The
high
price
and
limited
supply
of
natural
vanillin
have
necessitated
a
shift
towards
its
production
from
other
sources.
Hemicelluloses
are
readily
available
bulk
source
of
xylose
from
which
xylitol
and
furfural
can
be
derived.
Xylitol
used
instead
of
sucrose
in
food
as
a
sweetner,
has
odontological
applications
such
as
teeth
hardening,
rem-
ineralization,
and
as
an
antimicrobial
agent,
it
is
used
in
chewing
gum
and
toothpaste
formulations
(Roberto
et
al.,
2003).
The
yield
of
xylans
as
xylitol
by
chemical
means
is
only
about
5060%
making
xylitol
production
expen-
sive.
Various
bioconversion
methods,
therefore,
have
been
explored
for
the
production
of
xylitol
from
hemicellulose
using
microorganisms
or
their
enzymes
(Nigam
and
Singh,
1995).
Furfural
is
used
in
the
manufacture
of
furfural
phe-
nol
plastics,
varnishes
and
pesticides
(Montane
et
al.,
2002).
Over
200,000
tones
of
furfural
with
a
market
price
of
about
$1700
per
ton
is
annually
produced
(Zeitch,
2000).
Bio-Fuel
The
demand
for
ethanol
has
the
most
significant
market
where
ethanol
is
either
used
as
a
chemical
feedstock
or
as
an
octane
enhancer
or
petrol
additive.
Global
crude
oil
production
is
predicted
to
decline
from
25
billion
barrels
to
approximately
5
billion
barrels
in
2050
(Campbell
and
Laherrere,
1998).
They
produces
ethanol
from
the
fermen-
tation
of
cane
juice
in
Brazil
whereas
corn
is
used
in
the
USA.
In
the
US,
fuel
ethanol
has
been
used
in
gasohol
or
oxygenated
fuels
since
the
1980s.
These
gasoline
fuels
contain
up
to
10%
ethanol
by
volume
(Sun
and
Cheng,
2002).
It
is
estimated
that
4540
million
litres
of
ethanol
is
used
by
the
US
transportation
sector
and
that
this
amount
will
rise
phenomenally
since
the
US
automobile
manufac-
turers
plan
to
manufacture
a
significant
number
of
flexi-
fueled
engines
which
can
use
an
ethanol
blend
of
85%
ethanol
and
15%
gasoline
by
volume
(Sun
and
Cheng,
2002).
The
production
of
ethanol
from
sugars
or
starch
impacts
negatively
on
the
economics
of
the
process,
thus
making
ethanol
more
expensive
compared
with
fossil
fuels.
Hence
the
technological
focus
for
the
ethanol
pro-
duction
has
shifted
towards
the
utilization
of
residual
lignocellulosic
materials
to
have
lower
production costs.
Other
high
value
byproduct
of
products
such
as
organic
acids,
amino
acids,
vitamins
and
a
number
of
bacterial
and
fungal
polysaccharides
such
as
xanthan
are
produced
by
fermentation
using
glucose
as
the
base
substrate
but
theoretically
these
same
products
could
be
manufactured
from
"lignocellulosic
waste".
Production
of
extracellular
enzymes
by
fungi:
Extensive
studies
have
been
made
in
the
production
of
extracellular
enzymes
by
fungi.
Further
the
potential
applications
of
such
enzymes
in
the
bioconversion
of
lignocelluloses
to
economically
are
important
useful
products.
The
produc-
tion
of
the
cellulases,
microbial
protein
and
reducing
sug-
ars
(released)
were
also
submerged
culture
than
in
solid
state
fermentation
of
wheat
straw
by
Aspergillus
terreus
(Eyini
et
al.,
0000).
Cellulases
and
hemicellulases
have
numerous
applications
and
biotechnological
potential
for
various
industries
including
chemicals,
fuel,
food,
brew-
ery
and
wine,
animal
feed,
textile
and
laundry,
pulp,
paper
and
agriculture
(Bhat,
2000;
Sun
and
Cheng,
2002;
Wong
and
Saddler,
1992a,
b;
Beauchemin
et
al.,
2001,
2003).
It
is
estimated
that
approximately
20%
of
the
>1
billion
US
dollars
of
the
world's
sale
of
industrial
enzymes
consists
of
cellulases,
hemicellulases
,pectinases
and
the
world
market
for
industrial
enzymes
will
increase
in
the
range
of
1.72.0
billion
US
dollars
by
the
year
2005
(Bhat,
2000).
A
xylanase,
Novozyme
867,
has
shown
excellent
per-
formance
in
the
wheat
separation
process
(Christopherson
et
al.,
1997).
Hemicellulases
are
used
for
pulping
and
bleaching
in
the
pulp
and
paper
industry
where
they
are
used
to
modify
the
structure
of
xylan
and
glucomannan
in
pulp
fibres
to
enhance
chemical
delignification
(Suurnakki
et
al.,
1997).
A
patented
lignozyme
process
is
effective
in
delignifying
wood
in
a
pilot
pulp-
and
paper
process
(Call
and
Muck,
1997). In
bio-pulping
where
lignocellulytic
enzymes
were
used
the
following
was
achieved:
tensile,
tear
and
burst
indexes
of
the
resultant
paper
were
improved,
brightness
of
the
pulp
was
increased
and
an
improved
energy
saving
of
3038%
was
realized
(Scott
et
al.,
1998).
Laccases
can
degrade
a
wide
variety
of
synthetic
dyes
making
them
suitable
for
the
treatment
of
wastewa-
Bioconversion
of
Lignocellulose
Materials
161
ter
from
the
textile
industry
(Rosales
et
al.,
2002).
Organ-
isms
such
as
the
white
rot
fungi
producing
lignases
could
be
used
for
the
degradation
of
persistent
aromatic
pollut-
ants
such
as
dichlorophenol,
dinitrotoluene
and
anthracene
(Gold
and
Alic,
1993).
There
is
a
huge
potential
market
for
fibre-degrading
enzymes
for
the
animal
feed
industry
and
over
the
years
a
number
of
commercial
preparations
have
been
produced
(Beauchemin
et
al.,
2001,
2003).
The
use
of
fibre-degrad-
ing
enzymes
for
ruminants
such
as
cattle
and
sheep
for
improving
feed
utilization,
milk
yield
and
body
weight
gain
have
attracted
considerable
interest.
Steers
fed
with
an
enzymes
mixture
containing
xylanase
and
cellulase
showed
an
increased
live-weight
gain
of
approximately
3036%
(Beauchemin
et
al.,
1995).
In
dairy
cows
the
milk
yield
increased
in
the
range
4-16%
of
various
com-
mercial
fibrolytic
enzyme
treated
forages
(Beauchemin
et
al.,
2001).
Degradation
of
Lignocellulose:
Lignocellulose
consists
of
lignin,
hemicellulose
and
cellulose
and
compiled
from
Belles
et
al.
(1991);
Sun
and
Cheng
(2002)
shows
the
typical
compositions
of
the
three
components
in
various
lignocellulosic
materials.
Because
of
the
difficulty
in
dis-
solving
lignin
without
destroying
it
and
some
of
its
sub-
units,
its
exact
chemical
structure
is
difficult
to
ascertain.
In
general
lignin
contains
three
aromatic
alcohols
(Coniferyl
alcohol,
Sinapyl
and
p-coumaryl).
In
addition,
grass
and
dicot
lignin
also
contain
large
amounts
of
phenolic
acids
such
as
p-coumaric
and
ferulic
acid,
which
are
esterified
to
alcohol
groups
each
other
and
to
other
alcohols
such
as
sinapyl
and
p-coumaryl
alcohols.
Lignin
is
further
linked
to
both
hemicelluloses
and
cellulose
forming
a
physical
seal
around
the
latter
two
components
that
is
an
impene-
trable
barrier
preventing
penetration
of
solutions
and
enzymes.
Hemicellulose
macromolecules
are
often
polymers
of
pentoses
(xylose
and
arabinose),
hexoses
(mostly
man-
nose)
and
a
number
of
sugar,
acid
while,
cellulose
is
a
homogenous
polymer
of
glucose.
Of
the
three
components,
lignin
is
the
most
recalcitrant
to
degradation
whereas
cel-
lulose,
because
of
its
highly
ordered
crystalline
structure,
is
more
resistant
to
hydrolysis
than
hemicellulose.
Alka-
line
(Chahal,
1992)
and
acid
(Grethlein
and
Converse,
1991;
Nguyen,
1993)
hydrolysis
methods
have
been
used
to
degrade
lignocellulose.
Weak
acids
are
tend
to
remove
lignin
but
result
in
poor
hydrolysis
of
cellu-
lose
whereas
strong
acid
treatment
occur
under
rela-
tively
extreme
corrosive
conditions
at
high
temperature
and
pH
which
necessitate
the
use
of
expensive
equip-
ment.
Also,
unspecific
side
reactions
occur,
which
yield
non-specific
by-products
other
than
glucose,
promote
glu-
cose
degradation
and
therefore
reduce
its
yield.
Some
of
the
unspecific
products
can
be
deleterious
to
subsequent
fermentation
unless
removed.
There
are
also
environmen-
tal
concerns
associate
with
the
disposal
of
spent
acid
and
alkaline.
For
many
processes
enzymes
are
preferred
to
acid
or
alkaline
processes
since
they
are
specific
biocata-
lysts,
they
can
operate
under
much
milder
reaction
condi-
tions,and
they
do
not
produce
undesirable
products
and
are
environmentally
friendly.
Bioprocessing
of
lignocellulosic
materials:
Technologies
are
currently
available
for
all
steps
in
the
bioconversion
of
lignocelluloses
to
ethanol
and
other
chemical
products.
However,
these
technologies
must
be
improved
and
new
technologies
developed
to
produce
renewable
biofuel
and
other
byproducts
at
prices
which
can
compete
with
cur-
rent
production
costs.
The
feedstock
costs
can
be
mini-
mized
by
focusing
on
agricultural
residues
and
waste
materials
initially.
Other
process
steps,
which
are
particu-
larly
expensive,
include
pretreatments
to
improve
the
bio-
conversion,
the
production
of
enzymes
for
depolymerization
of
the
complex
raw
materials
and
capital
costs
associated
with
bioconversions.
In
general
the
technology
of
bioprocessing
of
raw
materials
or
their
constitutents
into
bioproducts
entails
three
steps,
process
design,
system
optimization
and
model
development.
Processing
involves
the
use
of
biocatalysts,
whole
microorganisms
or
their
organisms
to
synthesize
or
bioconvert
raw
materials
into
new
products;
recover/purify
such
bioproducts
and
subsequently
any
needed
downstream
modifications.
Solid
state
fermentation:
It
has
been
reported
that
the
solid
state
fermentation
(S
SF) is
an
attractive
alternative
process
to
produce
fungal
microbial
enzymes
using
ligno-
cellulosic
materials
from
agricultural
wastes
due
to
its
lower
capital
investment
and
lower
operating
cost
(Cha-
hal
et
al.,
1996;
Haltrich
et
al.,
1996;
Tech,
2000).
SSF
process
will
be
ideal
for
developing
countries.
Solid-state
fermentations
are
characterized
by
the
complete
or
almost
complete
absence
of
free
liquid
water,
which
is
essential
for
microbial
activities,
is
present
in
an
absorbed
or
in
complexed
status
form
with
the
solid
matrix
and
the
sub-
strate
(Canel
and
Moo-Young,
1980).
These
cultivation
conditions
are
especially
suitable
for
the
growth
of
fungi,
known
to
grow
at
relatively
low
water
activities.
As
the
microorganisms
in
SSF
grow
under
conditions
closer
to
their
natural
habitats
they
are
more
capable
of
producing
enzymes
and
metabolites
which
will
not
be
produced
or
will
be
produced
only
in
low
yield
in
submerge
conditions
(Tech,
2000).
SSF
are
practical
for
complex
substrates
including
agricultural,
forestry,
food-processing
residues
and
wastes which
are
used
as
carbon
sources
for
the
pro-
duction
of
lignocellulolytic
enzymes
(Haltrich
et
al.,
1996).
Compared
with
the
two-stage
hydrolysis
fermentation
pro-
cess
during
ethanol
production
from
lignocellulosics.
162
Pothiraj
et
al.
Sun
and
Cheng
(2002)
reported
that
SSF
has
the
fol-
lowing
advantages:
(1)
increase
in
hydrolysis
rate
by
con-
version
of
sugars that
inhibit
the
enzyme
(cellulose)
activity;
(2)
lower
enzyme
requirement;
(3)
higher
prod-
uct
yield;
(4)
lower
requirement
for
sterile
conditions
since
glucose
is
removed
immediately
and
ethanol
is
pro-
duced;
(5)
shorter
process
time;
and
(6)
less
reactor
vol-
ume.
In
a
recent
review
(Malherbe
and
Cloete,
2003)
reiterated
that
the
primary
objective
of
lignocellulose
treatment
by
the
various
industries
is
to
access
the
poten-
tial
of
the
cellulose
encrusted
by
lignin
within
the
ligno-
cellulose
matrix.
They
expressed
the
opinion
that
a
combination
of
SSF
technology
with
the
ability
of
an
appropriate
fungus
to
selectively
degrade
lignin
will
make
possible
industrial-scale
implementation
of
lignocellulose-
based
biotechnologies.
Like
all
technologies,
SSF
has
its
disadvantages
and
these
have
received
the
attention
by
Mudgett
(1986).
Prob-
lems
commonly
associated
with
scale-up,
biomass
growth
estimation
and
control
of
substrate
content.
However,
the
process
has
been
used
for
the
production
of
many
micro-
bial
products
and
the
engineering
aspects
and
the
scale-up
will
depend
on
bioreactor
design
and
operation
(Lonsane
et
al.,
1992).
A
recent
technical
report
in
2002
(http:/
www.lgu.umd.edu/outline.cfm)
on
"The
Science
and
Engi-
neering
for
a
bio-based
Industry
and
Economy"
has
ade-
quately
discussed
some
of
the
strategies
in
lignocellulose
bio-conversion
processes.
Other
lignocellulose
bioprocess-
ing
strategies
include
anaerobic
treatment,
composing,
production
of
single
cell
protein
for
ruminant
animal
feed-
ing
and
mushroom
cultivation.
These
processes
have
been
extensively
reviewed
(Smith
et
al.,
1987)
and
will
not
be
further
discussed
in
this
review.
Microorganisms
and
their
lignocellulytic
enzymes:
Var-
ious
lignolytic
waste
materials
such
as
hay,
barley
straw,
bagasse,
rye
straw,
newspaper,
saw
dust,
and
coconut
fibre
were
used
for
lignolytic
degradation
under
aerobic
diges-
tion.
Fungal
degradation
by
Phanerochaete
chrysospo-
rium
Polystictus
sanguineus,
Poria
subacida
and
Trametes
versicolor have
been
studied
in
fair
detail
(Kirk
and
Fenn,
1982).Palmer
and
Evans
(1983)
have
reported
that
few
actinomycetes
such
as
Streptomycetes
and
Nocardia
also
degrade
lignin.
A
diverse
spectrum
of
lignocellulolytic
microorgan-
isms,
mainly
fungi
(Baldrian
and
Gabriel,
2003;
Falcon
et
al.,
1995)
and
bacteria
(McCarthy,
1987;
Zimmermann,
1990;
Vicuna,
1988)
have
been
isolated
and
identified
over
the
years
and
this
list
still
continues
to
grow
rapidly.
Already
an
impressive
collection
of
more
than
14,000
fungi
which
were
active
against
cellulose
and
other
insol-
uble
fibres
were
collected
by
Mandels
and
Sternberg
(1976).
Despite
the
impressive
collection
of
lignocellu-
lolytic
microorganisms,
only
a
few
have
been
studied
extensively
and
mostly
Trichoderma
reesei
and
its
mutants
are
widely
employed
for
the
commercial
production
of
hemicellulases
and
cellulases
(Esterbauer
et
al.,
1991;
Jor-
gensen
et
al.,
2003;
Nieves
et
al.,
1998).
This
is
so,
partly
because
T
reesei
was
one
of
the
first
cellulolytic
organ-
isms
isolated
in
the
1950s
and
because
extensive
strain
improvement
and
screening
programs,
and
cellulose
indus-
trial
production
processes,
which
are
extremely
costly,
have
been
developed
over
the
years
in
several
countries.
T
reesei
might
be
a
good
producer
of
hemi-and
cellu-
loytic
enzymes
but
is
unable
to
degrade
lignin.
The
white-rot
fungi
belonging
to
the
basidimoycetes
are
the
most
efficient
and
extensive
lignin
degraders
(Akin
et
al.,
1995;
Gold
and
Alic,
1993)
with
P.chtysosporium
being
the
best-studied
lignin-degrading
fungus
producing
copious
amounts
of
a
unique
set
of
lignocellulytic
enzymes.
P
chrysosporium
has
drawn
considerable
attention
as
an
appropriate
host
for
the
production
of
lignin-degrading
enzymes
or
direct
application
in
lignocellulose
bioconver-
sion
processes
(Ruggeri
and
Sassi,
2003;
Bosco
et
al.,
1999).
Less
know,
white-rot
fungi
such
as
Daedalea
Phlebia
facicularia,
P
floridensis
and
P
radiate
have
been
found
to
selectively
degrade
lignin
in
wheat
straw
and
hold
out
prospects
for
bioconversion
biotech-
nology.
The
aim
is
just
to
remove
the
lignin
leaving
the
other
components
(Arora
et
al.,
2002).
Less
prolific
lig-
nin-degraders
among
bacteria
such
as
those
belonging
to
the
genera
Cellulomonas,
Pseudemonas
and
the
actino-
mycetes
Thermomonospora
and
Microbispora
and
bacteria
with
surface-bound
cellulose-complexes
such
as
Clostrid-
ium
thermocellum
and
Ruminococcus
are
beginning
to
receive
attention
as
representing
a
gene
pool
with
possible
unique
lignocellulase
engineering
(Vicuna,
1988;
McCarthy,
1987;
Miller
Jr.
et
al.,
1996;
Shen
et
al.,
1995;
Eveleigh,
1987;
Perestelo
et
al.,
1994).
Degradation
of
lignin
and
hemicellulose
was
also
achieved
by
Pal
et
al.
(1995)
during
the
cultivation
of
mushroom
Flammulina
velutipes
and
the
white
rot
fungus
Trametes
versicolor
on
sugarcane
bagasse
for
40
days.
Trametes
versicolor
produced
laccase
and
manganese-peroxidase
and
showed
a
simultaneous
degradation
of
lignin
and
holocel-
lulose.
However,
only
phenoloxidase
activity
was
found
with
Flammulina
velutipes,
which
exhibited
a
greater
reduction
in
the
ratio
of
weight
to
lignin
loss
than
Tram-
etes
versicolor.
They
also
proved
the
laccase
and
manga-
nese-peroxidase
activity
in
both
organisms.
The
maximum
laccase
activity
was
showed
by
Trametes
versicolor
on
5
th
day,
but
it
decreased
in
subsequent
days.
Flammulina
velutipes
showed
far
less
laccase
activity
than
Trametes
versicolor
under
the
assay
conditions
used.
A
bacterial
strain
of
the
Pseudomoncts
putida,
is
iso-
lated
from
decomposing
plant
material,
was
capable
of
degrading
lignin
related
compounds
and
also
observed
the
ability
of
this
bacterium
to
degrade
Kraft-lignin
and
radio-
Bioconversion
of
Lignocellulose
Materials
163
labelled
lignins.
Lignases
Fungi
can
breakdown
lignin
aerobically
through
the
use
of
a
family
of
extracellular
enzymes
collectively
termed
"lignases".
Two
families
of
lignolytic
enzymes
are
widely
considered
to
play
a
key
role
in
the
enzymatic
degrada-
tion:
phenol
oxidase
(laccase)
and
peroxidase.
(MnP)
(Krause
et
al.,
2003;
Malherbe
and
Cloete,
2003).
Other
enzymes
whose
roles
have
not
been
fully
elucidated
include
H,0,-producing
enzymes:
glyoxal
oxidase
(Kersten
and
Kirk,
1987),
glucose
oxidase
(Kelley
and
Reddy,
1986),
Veratryl
alcohol
oxidases
(Barbonnais
and
Paice,
1988),
methanol
oxidase
(Nishida
and
Eriksson,
1987)
and
oxido-
reductase
(Bao
and
Renganathan,
1991).
Enzymes
involved
in
lignin
breakdown
are
too
large
to
penetrate
the
unal-
tered
cell
wall
of
plants
so
the
question
arise,
how
to
lig-
nases
affect
lignin
and
biodegradation.
Suggestions
are
that
lignases
employ
low-molecular,
diffusible
reactive
com-
pounds
to
affect
initial
changes
to
the
lignin
substrate
(Call
and
Mucke,
1997)
Cellulases
In
most
lignocellulosic
materials,
cellulose
forms
the
major
part
of
the
three
components.
Cellulose
is
composed
of
insoluble,
linear
chains
of
fl-(1-4)-linked
glucose
units
with
an
average
degree
of
polymerization
of
about
10000
units
but
could
be
as
low
as
15
units
(Eveleigh,
1987).
Cellulases,
responsible
for
the
hydrolysis
of
cellulose,
are
composed
of
a
complex
mixture
of
proteins
with
differ-
ent
specificities
to
hydrolyze
glycosidic
bonds.
Cellulases
can
be
divided
into
three
major
enzyme
activity
classes
(Goyal
et
al.,
1991;
Robinovich
et
al.,
2002a,
b).
These
are
endoglucanases
or
endo-1,4-fl-glucanase,
cellobiohy-
drolase,
and
fl-glucosidase.
Endoglucanases,
often
called
carboxymethylcellulose
(CM)-cellulases,
are
proposed
to
initiate
attack
randomly
at
multiple
internal
sites
in
the
amorphous
regions
of
the
cellulose
fibre
opening-up
sites
for
subsequent
attack
by
the
cellobiohydrolases
(Wood,
1991).
Cellobiohydrolase,
often
called
an
exoglucanase,
is
the
major
component
of
the
fungal
cellulase
system
account-
ing
for
4070%
of
the
total
cellulase
proteins
and
can
hydrolyse
highly
crystalline
cellulose
(Esterbauer
et
al.,
1991).
A
cellulase
with
exo-and
endo-activities
from
Cal-
docellum
saccharolyticum
was
identified
(Saul
et
al.,
1990).
Xylanase
Hemicellulase
was
able
to
degraded
by
xylanase
enzyme.
Rabinovich
et
al.
(2002a)
and
Shallom
and
Shoham,
(2003)
present
recent
reviews
covering
the
types,
struc-
ture,
function,
classification
of
microbial
hemicellulases.
Hemicellulases
like
most
other
enzymes
which
hydrolyse
plant
cell
polysaccharides
are
multi-domain
proteins
(Hen-
rissat
and
Davies,
2000;
Prates
et
al.,
2001).
These
pro-
teins generally
contain
structurally
discrete
catalytic
and
non-catalytic
modules.
Xylan
is
the
most
abundant
hemi-
cellulose
and
xylanases
are
one
of
the
major
hemicellu-
lases
which
hydrolyse
the
/3-1,4
bond
in
the
xylan
backbone
hydrolyzed
into
single
xylose
units
by
fl-xylosi-
dase.
Conclusion
The
energy
and
environmental
crises
which
the
world
is
experiencing
is
forcing
us,
among
other
things,
to
re-eval-
uate
the
efficient
utilization
or
finding
alternative
uses
for
natural,
renewable
resources,
especially
organic
"waste",
using
clean
technologies.
The
same
strategic
imperatives,
economic
growth
and
developmental
issues
which
drove
Western
countries
research
into
lignocelluloses
since
the
1970's
are
of
even
greater
and
pressing
relevance
to
developing
countries.
Developing
countries
are
still
grap-
pling
with
socio-economic
issues
including
meeting
the
massive
energy-shortage
demands,
food
security
and
devel-
oping
technological
solutions
in
the
agriculture,
agro-pro-
cessing
and
other
related
manufacturing
sectors.
Ligno-
cellulose
biotechnology
offers
significant
opportunities
to
developing
countries
for
addressing
some
of
the
issues
highlighted
since
most
of
the
technology
is
based
on
the
utilization
of
readily
available
residual
plant
biomass
con-
sidered
as
"waste"
to
produce
numerous
value-added
prod-
ucts.
Brazil's
success
in
bio-fuel
is
often
a
show-case
of
but
one
example
of
the
economic
potential
for
develop-
ing
countries
in
the
area
of
lignocellulose
biotechnology.
On
the
other
hand
neglecting
this
technology
could
be
immensely
costly.
Already
patterns
of
production
and
trade
are
significantly
affected
by
the
emergence
of
biotechno-
logically
produced
goods
some
which
may
reduce
or
elimi-
nate
the
demand
by
Western
countries
for
agrarian
products
from
developing
countries.
For
example,
sugar
from
cane
can
be
replaced
by
enzyme
produced
sugar-syrups,
xyli-
tol,
glucose
and
fructose
sweeteners.
Lignocellulose
tech-
nology
may
be
transferred
to
developing
countries
but
at
exorbitant
prices
and
only
after
its
technological
and
busi-
ness
cycles
have
been
fully
exploited.
Lignocellulose
bio-
technology
from
a
capital
costs
investment
perspective
is
an
attractive
technology
for
developing
countries
since
its
biodegradation
could
follow
solid-state
fermentation
com-
parable
to
silage
or
mushroom
production,
thus
making
such
technology
suitable
for
farms
and
small
industrial
plants
without
the
need
for
large
engineering
infrastruc-
ture.
It
is
also
important
to
emphasize
that
in
order
for
lignocellulose
biotechnology
to
make
meaningful
impact
on
developing
countries;
suitable
bioconversion
processes
164
Pothiraj
et
al.
need
to
be
developed
on
a
much
wider
scale
and
these
countries
should
begin
to
pull
their
meager
resources
and
biological
science
expertise
in
a
cooperative
and
integrated
manner
towards
modern,
advance
genomics
and
proteom-
ics
technologies
for
identifying
novel
lignocellulolytic
enzymes
and
engineering
enzymes
with
improved
activi-
ties
suitable
for
industrial-scale
application.
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