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.

c)©

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).