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Microbial
Diversity Personal
Project
Isolation
of
CeluIolytic
Bacterium
using Turnip
as
Cellulose
Source Substrate
Jiancai
He
Dept.
of
Microbio.,
UMASS,
Amherst
Introduction
Interested
in cellulose
decomposition
and
in
the
microorganisms
that
produce
the relevant
enzyme systems has been stimulated by
a
desire for
a
greater understanding
of
one
of
the
most important
processes in
nature. This,
in
turn,
has been prompted
by the
need
to
optimize
exploitation
of
the
potential of cellulose
as
a
source of
energy
and
chemical
feedstocks
and
to improve
the
efficiencies
of
digestion
of fodder
by.
ruminants.
Cellulose
accounts , on average,
for
50
of the
dry
weight
of
plant biomass i t a lso
accounts for
about
50
of
the
dry weight of secondary
source
of
biomass,
such
as the
surpluses,
wastes of agricultural, forest, industrial
and
domestic origin. Cellulose
is a
homopolymer
consisting of
glucose
units joined
by f
1,4
bonds.
The disaccharide
cellobiose
is
rearded
as the
repeating unit in
cellulose
in
as
much
as each glucose units is
rotated
by
180 relative
to
its
neighbor.
Cellulolytic
microorganisms
are
ubiquitous
in nature,
and representative
species
are
found
in
many different
generation
and
in
a great variety of
environments
such
as
soils,
swamps,
seawater sediments,
cotton bales,
animal
gut
ect. Many enrichments
have been
done
for
cellulolytic
bacter ia and many
different
types of cellulolytic bacteria
have been isolated
and
pure
cultured. All of
these, enrichments
have
used
microcrystalline
cellulose,
carboxymethyl
cellulose CMC) or
other forms of
purified cellulose
as
carbon sources.
These celluloses
are quite
different
from
the
cellulose
found in many
vegetables or other
natural
sources.
In that natural,
cellulose
is
more
hydrated
than purified cellulose.
As
turnips contain
a
high
concentration of
hydrated
cellulose,
an enrichment
using turnip
slices
as the cellulose
source could turn
up
with
new
type
of cellulose degrading
bacteria.
In this project, I am using boiled turnip mash
as a
substrate
to
isolate cellulolytic bacteria.
Congo
Red stain
is
used
to
indicate cellulose
hydrolysis,
Cellulose
Azure
is
used
as
substrate
to do cellulose assay.
Materials and
Methods
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Enrichment, Culture conditions
and
media: Enrichment: The sources
of bacteria
were
the gut
of
a
terrestrial
snail
found in
Dr.
B.
Leadbetter
s
backyard and Eel Pond
sediment.
Boiled
and
autoclaved
turnip
slices
was used as
carbon
source.
Media:
All media
contained
g
/1)
K2HPO4
NH4C1
MgSO4.
71120 0.05
CaC12.
21120 0.02
add water
to
1000
ml .
In
enrichment
broth, turnip
slices
were made
by
boiling
times and
then
autoclaved;
in th e
isolation
media, turnip
paste
were
made
by
boiling
turnip slices
35
times, throw
the
water each time, then
use
blend mash
the
turnip into very f ine
paste
solution;
Washed Agar
were made
by
Add
25
g
Agar
to
51
distilled
water,
stirred
for hour
then
replace water for
one cycle ,
generally
5
6 cycles,
original
25
g
agar used for 1000
ml media. Cellobiose 2
mg/mi f inal concentration), cellulose
ball
milled filter paper, mg
/
ml),
CMC 1 mg
/ml)
and xylan 1 mg
/
ml)
agar
plate
were
also
made
by
adding
these sugars
to th e
base
media
given
above.
Cellobiose, glucose
and
CMC broth media were made
by
adding those
sugar solution
to the
autoclaved
base
media before
inoculation.
10
cellobiose
and
glucose stock solutions were filter sterilized
Culture Conditions: Aerobic isolates were
cultured
in
37C
incubator. naerobic isolate
was
culture
in room
temperature under N2
C02 in
the
anaerobic chamber.
Congo Red
test: Follow protocol according
to
Teather, after appropriate growth period,
flood
the
plate with
the
Congo Red Solution
1
mg
/
m l in water)
for 15
minutes.
For
CMC, pour off Congo Red and flood plate with M Na l
for
15
minutes. Visualized
zones
of
hydrolysis
can be
stabilized for
at
least 2 weeks
by
flooding the
agar
with
M
HC1,
which
will
change
the dye
color
to
blue
and
inhibit further enzyme
activity.
Oxidase test:
Use
spot
test oxidase reagent form Difco
lab.
Pick
up
colonies from
plate,
put them
on
the filter
paper, d rop one o r
two
drops
of
oxidase
reagent
to
the colonies,
check blue color
for
positive
result
after
5
minutes.
Colorless
is
negative.
Electron
microscopy:
Cell
were negatively stained
for
microscopy.
arbon coated
grids were
used
to
absorb cells
from
well
prepared bacterial
cell
suspension
for 2 minutes.
Uranyl
acetate
solution 1 wt
/
vol, pH 4 .5 ) was used. Negatively stained
preparations
was
examined
using
a
Zeiss 1OCA transmission electron microscope.
In Situ 16s
RNA
Hybridization:
Follow Dr . Sandra A. Nierzwicki Bauers
Handout.
Cellulose
Assay:
Use
cellulose Azure
as a
substrate.
Suspend 100 mg of cellulose
zure in
lOmi
phosphate
buffer
with
no
cellulose.
In
microcentrfuge tube, put
ml
cellulose Azure solution
and
ml
cell
culture incubate for different period time
at room
temperature. entrifuge
for
3
4 minutes,
measure supernatant absorption
at 570
nm ,
DH2O
and
cellulose were
used as
negative and positive controls.
Results and Discussions
J-f
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nr
ich
m
en
t
a
nd
mor
phol
ogy
Enri
chm
ents
we
re strea
ked
o
n the
is
olat
ion
pl
ate
p
ut
u
nde
r
ae
robi
c
and a
naer
obic
con
ditio
ns.
there were
on g
rowt
h
f
rom
s
ea
sedi
men
t From
snail
gut,
thr
ee
aero
bic
an
d one
anae
robi
c
strai
n
w
ere
isola
ted
Co
lony
a
nd M
o
rp
ho
lo
gy
is
olate
s
colo
nies
mo
rpho
logy
con
go
oxid
ase Gr
am
ae
robi
c
or
r
ed
te
st stain
ana
erob
ic
tes
t
larg
e w
hit
e,
s
ingl
e,
thin
but
a
naer
obic
v
ery
co
nvex
l
ong
c
ells
,
term
ina
l
end
osp
ore
fo
rme
r
S
w
hite
,
fiat
,
lar
ge
rod
form
?
ae
robi
c
ir
regu
lar
fi
lame
nts,
e
dge,
lo
ok
m
iddle
li
ke
my
celi
al
endo
spor
e
c
olon
ies
fo
rme
r,
gl
iding
1S
3
tin
y,
w
h
ite
rod
or
cocc
i,
aer
obic
conv
ex
non
mot
ile,
si
ngle
or pair
,
no
nend
osp
ore
form
er
1
S4
large
.wh
ite
sho
rt
r
od,
aero
bic
gl
isten
ing,
mo
tile,
sing
le
re
gula
r
edg
e no
en
dosp
ore
F
rom
t
he
turn
ip
pl
ate,
I
S
an
d 1S
2
ga
ve
c
lear
z
ones
arou
nd
the
col
onie
s,
so
th
is
tw
o
strai
ns
w
ere
cho
sen
to
do
furt
her
studi
es.
Ph
ysio
logy
Inoc
ulat
e
IS
an
d 1S2
on diff
eren
t
pl
ates
w
hich
co
ntai
n
diff
eren
t
ca
rbon
sou
rce
CM
C
c
ello
bios
ce
llulo
se
xy
lan
unw
ashe
d
w
ashe
d
a
gar
ag
ar
s
1S2
:
g
row
v
ery
w
ell
:
gr
ow
w
ell
n
o gro
wth
1S
2 w
as t
aken
fro
m
plat
e
an
d ino
cula
ted
to
li
quid
broth
m
edi
um
u
nde
r roo
m
tem
per
atur
e,
and th
ere
w
ere
no
gr
owt
h.
But if
add
0.01
yeas
t e
xtrac
t,
all o
f
the
m
gro
w
rap
idly
,
the
n
egat
ive
co
ntro
l
whic
h
h
as n
o ca
rbon
sou
rce
also
gr
ow, b
ut n
ot
a
s
turbi
d
a
s
o
ther
tu
bes
wi
th
car b
on
so
urce in
it
So
1S2
m
ay
ne
ed
som
e
grow
th
fa
ctor
fro
m
yea
st extr
act.
16
s
ri
bo
so
in
e
R
NA
h
yb
rid
iz
at
ion
a
n
d
ce
llu
la
se as
sa
y
dif
feren
t
rR
NA
prob
es
w
ere u
sed
to
do the hy
brid
izati
on.
F
or
t
he
ana
erob
ic
is
olati
on
I
s
1
al
l
the p
robe
s
are
ne
gativ
e,
even universal probe. the reason could be due
to
the old
cultu
re
wh
ich
co
ntai
n
all
e
ndo
spor
es
ins
tead
of ve
gita
tive
ce
lls. I
ts h
ard
for
the
p
robe
s
to
-
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4/8
get
i
nto
the
ce
ll an
d
g
ave
the
si
gnal
s.
Fo
r
1S2
,
p
rime A
w
hich
is
un
iver
sal
pr
obe w
as
t
he
only
p
ositi
ve
r
esu l
t.
ma
ybe
this
or
ga ni
sm
is
be ye
nd
th
e
det
ec t ran
ge of
al
l
prob
e s. Bes
ide s
,
the
re
ar
e
som
e imm
atu
re
asp e
sts
of 16s
rRN
A
tec
hen
ics
O
nly
1S2
was
perf
orme
d
of
ce
llula
se a
ssa y
. the
mec
heni
sm for th
is
ass a
y is cel
lulos
e
a
zure
whi
ch
is
ce l
lulos
e deriv
tive
that
h
as
azu
re b
ind i
ng to ce
llulo
se m on
om e
r, w
hen ce
llulo
se
azure
is
degradated
by
cellu lase,
the
product
show optical
absorption
at
570nm. This assay
too
k v
ery lon
g ti
me ,
the po
sitiv
e c
ont r
ol
turn
pin
k
afte
r 4
days 86 hou
rs .
the
assa
y wa s
ca
rried
ou
t
in
room
tem
pra
ture, the
p
ositi
ve co
ntrow
as m
ade
b
y
addi
ng 25
mg
puri
fed
ce l
lulas
e
to
m
l
ph
osph
ate
b
uffe
r. Dis
tille
d
wa
ter
w
as u
sed
as
n
eg tiv
e
co
ntro
l,
th
e sa
mpl
e
is m
ore lik
e
neg
tive
co
ntrol
.
ov
eral
l, th
is
ass
ay
w
as
a ver
y
r
ough
cellu
lase
as
say.
An
d
mo
re
this
a
ssay wa
s de
signe
d fo
r com
ple
te
cellu
lase sy
stem
. 1S
2
p
rob
ably
don
t
po
ssos
e
a
com
plet
e cellu
lase
sys
tem
since
it
d
idn
t
g
row
on
cell
ulas
e
pla
te, but it
ca
n
u
se
ce
llobi
ose
a
nd CM
C,
so
it
may
pro
duce
end
oglu
cana
ses
or glu
cosi
dase
s no
t the
com
plet
e
sy
stem
,
th
ose ba
cter
a,
as s
ugge
sted b
y Beg
uin , c
alled
pse
udo
ce llu
loly
tic in
co nt
rast
to
f
ew
b
ac te
ria
t
ha t
syn
the s
ize t
he c
omp
le te en
zym
e sy
stem th
at
c
ould
res
ult
in
ex
ten s
ive
h
yd r
olysi
s o
f
the
cry
stall
ine mat
er ial fo
und in
na tu
re, wh
ich i
s ca
lled tru
e ce l
lu lo l
ytic
.
It
is
p
ossib
le
t
hat
IS co
uld
be
true ce
llulolytic because it grow
on
cellulose plate.
Further
s
tud ie
s ha v
e
t
o
be done
to
defi
ne thi
s
ba
cter
ium.
A
ckno
wled
gme
nt:
I
than
k
Dr.
A
Sa
lyer
s for
he
r dir e
ction a
nd d
isscu
sion
and
l
ie
Hu
ang
f
or t
ypin
g
and
enco
urag
eme
nt
e
feren
e
Su s
an B.
Les
chin
e 1
995
,
Cell
ulos
e De
grad
ation in an
aero
bic
en
viro
nm e
nt.
An
nu.
Re
v.
Mic
robio
l.
19
95 .4
9:3 9
9 42
6
A
lber
t Balo
ws
et al 19
92 , T
he
Pro
kary
otes
2
nd Ed i
tion,
Sp rin
ger
Ver
lag
p
age 460
-5 16
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Acknowledgment:
I
thank Joel
for
his
direction
and disscusion and
Jie
Huang
for
her
typing
and encouragement.
Reference:
Susan
E.
Loure
Michael K. The odorou
Anthony
P.
J.
Trinci
and Robert B.
Hespell
1985 ,
Growth
of
Anaerobic
Rumen fungi
on
Defined and Semidefined
Media lacking
Rumen fluid.
Journal
of
General Microbiology
1985 ; 131,
2225 2229.
M.
J. Teunissen
A.
A.
M.
Smiths,
H.
J.
M. opden
Camp,
J.
H.
J. Huris int
Veld G.
D.
Vogels,
1991 , Fermentation
of cellulose
and production
of cellulolytic and xylanolytic
enzymes
by
anaerobic
fungi
from ruminant
and non-ruminent
herbivores.
Arch
Microbial
1991 ;
156:
290 296.
Susan
B.
Leschine
1995 ,
Cellulose
Degradation in anaerobic environment.
Annu.Rev.Microbiol. 1995.49:399 426
Teunissen
MJ,
Kets
EP ,
et.
al
1992 , Effect
of
coculture
of
anaerobic fungi
isolated
from
ruminants
and nonruminants
with methanogenic
bacteria on cellulolytic
and xyanolytic
enzyme
activities,
Arch
Microbial
1992 ;
157 2 :
176-182.