ilka faath various habitats germany microbial diversity 1994 · isolation of chitin degrading...
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Independent Student Research Project
MICROBIAL DIVERSITY 1994
MBL, WOODS HOLE
ISOLATION OF CHITIN DEGRADING BACTERIA FROM
VARIOUS HABITATS
ILKA FAATH
UNIVERSITY OF BONN
GERMANY
Introduction
Next to cellulose chitin is the most abundant biopolymer found in nature. Its mineralization is
mainly carried out by microbes. Chitin presents the sole source of carbon and nitrogen for many
bacteria. Chitinoclastic microorganisms have been isolated from numerous environments including
marine environments, exoskeletons of crustaceans and insects, intestines of vertebrates and
invertebrates, sands, muds, garden soils and lake waters. Most notable among the chitin degrading
procaryotes are the gliding bacteria, pseudomonads, vibrios, Photobacteriwn , enteric bacteria,
actinomycetes, bacilli and clostridia (Godday, G.W., 1990). Considering the variety of habitats in
which chitin degrading bacteria can be encountered, an isolation of different chitin degrading
bacteria and a subsequent characterization of them was attempted in this study.
Chitin degraders, especially in aquatic environments, might attach to the chitin source in order to
optimize the uptake of soluble products released during chitin hydrolysis. During this project
scanning electron microscopiè studies of the carapace ofLimutu.spolyphemus were carried out in
order to investigate the possibility of a specific attachment of chitin degrading bacteria to the
carapace.
Methods
Preparation of acid reprecinitated chitin (Hsu. S.C. and J.L. Lockwood. l975
40 g commercial chitin (USB Micro) was suspended in 600 ml 32% HC1. Within 30 minutes a thin
dark greyish colloidal solution was obtained. The solution was poured in 2 lice cold water
resulting in a pure white fluffy chitin precipitate. The suspension was centrifuged 5,000 rpm at 4°C
for 10 minutes. The pellets were washed and centrifuged again. Dialysis tubes (MWCO 25,000)
C were filled with the chitin pellets. The chitin was dialysed against tap water for 36 hours. During
the following 12 hours a dialysis against distilled water was carried out. The pH was determined as
4.8 (should be above 4.5) and adjusted to 7.2. The concentration of the chitin suspension was
adjusted to 5% with distilled water. After dividing the suspension in 100 ml aliquots the chitin
suspension was autoclaved.
Medium
Marine and freshwater/soil two layered agar plates were used for isolation of chitin degrading
bacteria from the different environments. The medium composition is presented in table 1. The
chitin suspension was incorporated into the overlay to a final concentration of 0.5%. Afterautoclaving the anaerobic medium was cooled under N2,C02 to a temperature of 55°C. Six vitamin
solution, vitamin B12 solution, NaHCO3 (1M, pH 7.0), Na2HPO4 and cystein were added. For
both aerobic and anaerobic media chitin was added after autoclaving.
The anaerobic medium was transfered into the glove box where the plates were poured.
Inocula sources
Marine sources used for the isolation of chitin degrading bacteria included the carapace of the horse
shoe crab (Linwius polyphemus), water and sediment from Sippewissett Salt Marsh as well as
water and sediment from Oyster Pond. Freshwater samples were taken from water and sediment of
School Street Marsh and Cedar Swamp. Soil samples were collected from Bell Tower.
Inoculation and incubation
Serial dilutions were carried out (i01, io-2, io-3, 10). An aliquot of each dilution step wasstreaked out on either freshwater/soil or marine agar plates under aerobic and anaerobic conditions.Incubation was carried out at room temperawre until clearing zones around single colonies beáamevisible. Positive colonies were restreaked to obtain pure cultures.For detection of bioluminescent chitin degrading bacteria, plates were observed in the dark.
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Tab
le1
Med
ium
com
posi
tion
for
chit
indeg
radin
gm
arin
e,fr
esh
wat
eran
dso
ilm
icro
orga
nism
scu
ltiv
ated
under
aero
bic
and
anae
robic
cond
itio
ns
Med
ium
com
pone
ntm
arin
efr
eshw
ater
/soi
l(f
or10
00m
l)ba
seov
erla
yba
seov
erla
y
NaC
125
.0g
25.0
g-
-
Mg
Qx6H
O3.
0g
3.0
g-
-
KC
10.
5g
0.5
g-
-
Na2
804
0.1
g0.
1g
--
NH
4C1
0.3
g0.
3g
--
CaC
12x2
H2O
0.0.
1g
0.01
g-
-
MgS
O4x
7H2O
1.0
g1.
0g
Na2
HPO
40.
25g
0.25
g-
-
K2H
PO4
-0.
2g
NaH
CO
3(1
M,
pH7.
0)30
.0m
l30
.0m
l-
-
Na-
Ace
tat
0.01
g-
Yea
stex
trac
t0.
1g
0.1
g0.
5g
-
Cas
iton
01
g01
g1.
0g
-
Six
vita
min
solu
tion
1.0
ml
1.0
ml
1.0
ml
1.0
ml
Vit
anii
nBl2
solu
tion
1.0
ml
1.0
ml
1.0
ml
1.0
ml
SLIO
1.0
nil
1.0
ml
1.0
ml
1.0
ml
Chi
tin5
%10
0.0
ml
100.
0m
lR
esaz
urin
o.1%
1.0
ml
1.0
ml
1.0
ml
1.0
ml
(for
anae
robi
cpl
ates
)C
yste
in5%
(pH
7.0)
10.0
ml
10.0
ml
10.0
ml
10.0
ml
(for
anae
robi
cpl
ates
)Se
leni
Tun
gste
nso
lutio
n1.
0m
l1.
0m
l1.
0m
l1.
0m
l(0
.1m
M,f
oran
aero
bic
plat
es)
Aga
r15
.0g
15.0
g15
.0g
15.0
g
pH7.
5pH
7.5
pH7.
5pH
7.5
Scanning electron microscopy
Fixation:
Carapace pieces from Limulus polyphemus were taken from different parts of the crustacean body
and fixed in 4% glutaraldehyde for 2 hours 30 minutes at room temperature.
Washing:
The samples were washed 5 mm in seawater.
Dehydration:
The samples were transferred to 70% EtOH and stored at 4°C over night. Further dehydration steps
were carried out in 80% EtOH (lx) and 90% EtOH (lx) for 5 mm and in 95% EtOH (2x) as well
as 100% EtOH (3x) for 20 mm at room temperature.
Critical point drying:
The dehydrated samples were transferred to the critical point dryer and dried.
Coating:
The dried samples were coated with a Pd/Au layer in the automatic sputter coating apparatus.
The samples were subsequently observed with the SEM.
Results
CClearing zones around colonies were observed after different time intervals depending on the
sampling site and on the presence or absence of 02 during cultivation (table 2).
Source Aerobic Anaerobic
marine
Limulus polyphemus after 3.5 days after 5.5 days
Sippewissett salt marsh after 5 days after 6 days
Oyster pond after 5 days after 6 days
freshwater
School steet marsh after 2 days after 2 days
Cedar swamp after 3 days after 4 days
2il
Bell Tower after 5 days after 6 days
Table 2
Different isolates from soil, niarine and freshwater environments were characterized by cell
morphology, motility, gram stain, their ability for aerobic or anaerobic growth, their pigmentation
and their ability to form endospores. Furthermore a bioluminescent, chitin degrading bacterium
was isolated from Sippewissett Salt Marsh. (Table 3,4 and 5).
Table
3M
arinechitin
degradingisolates
onag
arplates
IsolateSource
Gram
stain0
,M
otilityM
orphologyPigm
entB
ioluminescence
CD
1L
imulus
-fak.
+rod
--
polyphem
usanaerob
CD
2O
ysterpond-
+÷
rod+
-
(1)3O
ysterpond
-+
+vibrin
-
CD
4O
ysterpond-
-N
Drod
--
CD
5Sippew
issett-
++
rod-
÷saltm
arsh
CD
6Sippew
issett-
-N
Drod
--
salt marsh
0o
Table
4F
reshwater
chitindegrading
isolateson
agar
plates
IsolateSource
Gram
stainO
Motility
Morohology
Pigment
EndosD
oreform
ing
CD
7School
street-
fat
+rod
-
marsh
anaerob
CD
8School
streetgram
variable+
-rod
-+
marsh
CD
9C
edarswam
p-
-+
rod-
-
CD
1OC
edarsw
amp
-+
+vibrio
-
CD
11C
edarswam
p+
-N
Drod
-+
Table
5Soil
chitindegrading
isolateson
agar
plates
IsolateSource
Gram
stainO
Motility
Morphology
Pigment
Endospore
forming
Dl2
Befitow
er-
+-
rod4
-
CD
13B
eiltower
-+
-rod
+-
CD
14B
eiltower
--
ND
rod-
-
00
C) Inocula from several parts of the horse shoe crab carapace were taken. However the findings
suggest that only one bacterium was isolated from all horse shoe crab samples under aerobic andanaerobic conditions. SEM studies showed that large areas of the carapace were predominantlycolonized by one bacterial morphotype. These short rods are polarly attached to the surface of thecarapace (appendix).
Discussion
Chitin degraders were isolated from different habitats and compared. Differences in the rate of
chitin degradation as indicated by the appearance of clearing zones around single colonies on agarplates could be stated for the various isolates.
Three main conclusions can be drawn from the data presented in table 2:
1. Chitin degradation took place at a slower rate under anaerobic conditions (except School Street
Marsh isolates).
2. Freshwater chitin degrading bacteria (especially from School Street Marsh) degraded chitinfaster than the observed marine and soil chitin degraders.
3. Freshwater chitin degraders from School Street Marsh degraded chitin at the same rate underaerobic and anaerobic conditions.
Only one chitin degrading bacterium was isolated from the horse shoe crab carapace. SEM studiesrevealed a predominating bacterial morphotype on large areas of the carapace. These rods showed apolar attachment. As demonstrated for Vibrio hareyi a specific binding process could be involvedin the attachment (Montgomery, M.T. and D.L. Kirchman, 1993).
References
Gooday G.W. 1990. The ecology of chitin decomposition. Adv. Microb. Ecol. 11: 387 - 430.
Hsu, S.C. and J.L. Lockwood. 1975. Powdered chitin agar as a selective medium for enumerationof actinomycetes in water and soil. AppI. Microbiol. 29: 422 - 426.
Montgomery, M. T.and D. L. Kirchman. 1993. Role of chitin-binding proteins in the specificattachment of the marine bacterium Vibrio harveyi to chitin. AppI. Environ. Microbiol. 59: 373 -
379.
1)
2)
Colonization of the carapace of Limuluspotyphemus by poiariy attached rods (1) Overview (x3300) and (2) in detail (X 10,000).