Independent Student Research Project MICROBIAL DIVERSITY 1994 MBL, WOODS HOLE ILKA FAATH UNIVERSITY OF BONN GERMANY

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1 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. l 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

2 C for 10 minutes. The pellets were washed and centrifuged again. Dialysis tubes (MWCO 25,000) 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%. After autoclaving 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, io2, io3, 10). An aliquot of each dilution step was streaked 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 beáame visible. Positive colonies were restreaked to obtain pure cultures. For detection of bioluminescent chitin degrading bacteria, plates were observed in the dark.

3 c) ph 7.5 ph 7.5 ph 7.5 ph 7.5 Table 1 Medium composition for chitin degrading marine, freshwater and soil microorganisms cultivated under aerobic and anaerobic conditions Medium component marine freshwater/soil (for 1000 ml) base overlay base overlay NaC g 25.0 g MgQx6HO 3.0 g 3.0 g KC1 0.5 g 0.5 g Na g 0.1 g NH4C1 0.3 g 0.3 g CaC12x2H2O g 0.01 g MgSO4x7H2O 1.0 g 1.0 g Na2HPO g 0.25 g K2HPO4 NaHCO3 (1M, ph 7.0) 30.0 ml 30.0 ml NaAcetat 0.01 g 0.2 g Yeastextract 0.1 g 0.1 g 0.5 g Casiton 01 g 01 g 1.0 g Sixvitaminsolution 1.0 ml 1.0 ml 1.0 ml 1.0 ml VitaniinBl2solution 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 Chitin5% ml ml Resazurino.1% 1.0 ml 1.0 ml 1.0 ml 1.0 ml (for anaerobic plates) Cystein 5% (ph 7.0) 10.0 ml 10.0 ml 10.0 ml 10.0 ml (for anaerobic plates) SeleniTungsten solution 1.0 ml 1.0 ml 1.0 ml 1.0 ml (0.1 mm, for anaerobic plates) Agar 15.0 g 15.0 g 15.0 g 15.0 g

4 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.

5 C Results Clearing 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).

6 0 o CD1 Limulus fak. + rod polyp hem us anaerob (1)3 Oyster pond + + vibrin CD4 Oysterpond ND rod CD5 Sippewissett + + rod salt marsh CD6 Sippewissett ND rod salt marsh Table 3 Marine chitin degrading isolates on agar plates Isolate Source Gramstain 0, Motility Morphology Pigment Bioluminescence CD2 Oysterpond + rod +

7 0 0 CD7 School street fat marsh anaerob CD8 School street gram variable + marsh rod + CD9 Cedarswamp + rod CD1O Cedar swamp + + vibrio CD11 Cedarswamp + ND rod + + rod 4 CD14 Beiltower ND rod Table 4 Freshwater chitin degrading isolates on agar plates Isolate Source Gram stain O Motility Morohology Pigment EndosDore forming Table 5 Soil chitin degrading isolates on agar plates Isolate Source Gram stain O Motility Morphology Pigment Endospore forming CD13 Beiltower + rod + Dl2 Befitower + rod

8 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 and anaerobic conditions. SEM studies showed that large areas of the carapace were predominantly colonized by one bacterial morphotype. These short rods are polarly attached to the surface of the carapace (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 agar plates 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 chitin faster than the observed marine and soil chitin degraders. 3. Freshwater chitin degraders from School Street Marsh degraded chitin at the same rate under aerobic and anaerobic conditions. Only one chitin degrading bacterium was isolated from the horse shoe crab carapace. SEM studies revealed a predominating bacterial morphotype on large areas of the carapace. These rods showed a polar attachment. As demonstrated for Vibrio hareyi in the attachment (Montgomery, M.T. and D.L. Kirchman, 1993). a specific binding process could be involved References Gooday G.W The ecology of chitin decomposition. Adv. Microb. Ecol. 11: Hsu, S.C. and J.L. Lockwood Powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. AppI. Microbiol. 29: Montgomery, M. T.and D. L. Kirchman Role of chitinbinding proteins in the specific attachment of the marine bacterium Vibrio harveyi to chitin. AppI. Environ. Microbiol. 59:

9 3300) and (2) in detail (X 10,000). Colonization of the carapace of Limuluspotyphemus by poiariy attached rods (1) Overview (x 1) 2)