Polymer-degrading bacteria associated with thebrown algae Fucus spp.

Elke Allers

Carried out as an Individual Project within theMicrobial Diversity Course 2006

at the Marine Biological Laboratory in Woods Hole

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Abstract

The attempt to isolate polymer-degrading bacteria from the brown algae Fucus spp.

resulted in 20 strains which, according to 16S rRNA gene sequence analysis, were

members of the Alpha- and Gammaproteobacteria and the Bacteroidetes. The isolate

collection was dominated by strains closely related to Vibrio alginolyticus. In order to

compare growth kinetics and C-source utilization 5 strains were chosen for further

analysis. The gammaproteobacterial strains Pseudoalteromonas sp., Alteromonas sp.

and a relative of Cellvibrio reached higher cell yields than the Bacteroidetes strains

Cellulophaga sp. 2 and 5c, no matter whether they were grown on polysaccharides or

simple sugars. Pseudoalteromonas sp. was identified as the most successful utilizer of

fucoidan (an algal-derived polysaccharide), had the shortest lag phase (3 h) and the

shortest doubling time (0.3 h on Glucose). In contrast, both Cellulophaga strains

displayed lag phases of at least 12 h on any substrate studied, and moreover did not

start doubling at all on fucoidan before the experiment was over. The results suggest,

that within the strains isolated two different ecological strategies have been

observed: The gammaproteobacterial strains being ready to take available C-sources,

and the Cellulophaga strains rather taking time to adapt.

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1. Introduction

The Cytophagales are a very diverse group within the Bacteroidetes. They inhabit

many different types of habitats, ranging from soil to water, from fresh water to sea

water, from animal dung to decaying plant-material, from free-living to an attached

live style. Especially for the marine world they are known for being able to degrade

complex substrates like agar (Balows et al. 1992).

Another natural and in the same time biotechnically relevant polymer is fucoidan. It is

derived from seaweed, e.g. Fucus spp., and consists a fucose backbone. Since it is a

natural compound, one would expect bacteria to exist which can degrade and utilize

the fucoidan. These bacteria and their fucanase activity would provide a source of the

degrading enzyme and thereby a way to produce bioactive compounds, which are e.g.

involved in blocking infections by certain viruses like herpes simplex and HIV

(Descamps et al. 2006).

The idea of this study was to attempt the isolation of polymer-degrading bacteria and

to compare their metabolic traits in terms of polymer and simple sugar utilization.

2. Material & Methods

Isolation. The algal thalli were all collected at the same day, either directly at

Garbage Beach, Woods Hole (decaying material; anaerobic and aerobic) or further out

at the dock (living material; aerobic). Each thallus was put into sterile Seawater Base

(see below) and blended until everything was a slurry. One hundred µl of this slurry

were plated onto spread-plates either pure or in dilutions 1:10, 1:100, and 1:1000. As

soon as growth became apparent on the plates, colonies were re-streaked for

isolation. They were transferred at least 3 times before they were considered a pure

isolate.

The media. 1 x Seawater Base was the basis of all media used in this study (course

hand out 2006). According to the Modular Medium Approach presented during the

course (J. Leadbetter) nutrients and substrates were added (see Tab. Aa.1 and Aa.2)

adjusted to the needs. The isolation was carried out on plates, whereas growth

experiments were all done in liquid culture in tubes set up on a shaker at 30°C. 5 ml

of the Medium basis were dispensed into culture tubes. Agar and fucoidan were added

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to the tubes before autoclaving, the simple sugars were sterile-filtered through 0.2 µ

and then added to the already autoclaved medium.

Colony-PCR. The analysis of the 16S rRNA gene sequences of all isolates was carried

out according to the course hand out MD2006 (chapter 11. Phylogentic analysis of

bacterial isolates, see Appendix). Colonies were either picked directly into the PCR-

mix or into 10 µl of PCR water. The latter was vortexed and 1 µl was added to the

PCR. For full sequences 3 different primers were run in the sequencing reaction for

the same PCR product: 8F, 519F and 1492R. Sequence data was checked for next

relatives using the Blastn tool (www.ncbi.nlm.nih.gov/BLAST), and phylogenetic

analysis was done within the software package ARB.

Growth kinetics. Growth in general and the exponential growth phase in particular

were monitored by OD600 measurements on a photometer against a medium blank.

Doubling times were calculated according to the following equation: Td = ln2/µ,

where Td is the doubling time, and µ is the slope of the trend line added to a

density/time plot in exponential phase.

Degradation and utilization of polymers and utilization of sugars. HPLC analysis was

carried out according to the course hand out (see Appendix).

CARD-FISH on environmental samples. The bacteria present in the algal slurry were

studied by applying CARD-FISH (catalyzed reporter deposition fluorescence in situ

hybridization) according to Pernthaler et al. (2004). The following probes were used:

EUBI-III (Amann et al. 1990, Daims et al. 1999) to test the overall detection rate,

CF319a (Manz et al. 1996) specific for Cytophacga-Flavobacteria, ALT1413 (Eilers et

al. 2000) targeting representatives of the Alteromonadales. A nonsense probe NON338

(Wallner et al. 1993) was applied as a control for specificity.

3. Results

During this study 21 bacterial strains were isolated and identified by 16S rRNA gene

sequence analysis (Tab. Aa.3, see Appendix).

Most of the isolates belong to the Gammaproteobacteria and within these they find

their closest relatives in representatives of the Alteromonas, Pseudoalteromonas and

Vibrio. One isolate showed a similarity of 100% to the Alphaproteobacterium Stappia

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aggregata. Another two isolates were identified as close relatives of Cellulophaga

lytica. Five isolates were picked for further analysis. They are indicated in bold.

Microscopic images are provided in the Appendix, too (Fig. Ab.1).

All isolates displayed specific substrate usage. In terms of utilization of the tested

polysaccharides, it became obvious that agar as a C-source as opposed to fucoidan

leads to higher yields in cell density or to growth, at all. Generally, all strains isolated

in this study grew well with agar as sole carbon source. However, there were distinct

differences in the isolates’ affinity to this substrate. Pseudoalteromonas sp. was the

fasted to respond with growth and reached alltogether with the two other

gammaproteobacterial isolates the highest yields ranging between 0.4 and 0.5 OD600

(Tab. Aa.4 and Fig. Ab.2, Appendix). On the contrary, the Cellulophaga isolates

reached densities of ~0.25 and ~0.1, respectively. Moreover, their lag phase as well as

the one of Alteromonas sp. lasted for at least 12 hours.

Unfortunately, at this point of time the HPLC data has not been analyzed yet.

By applying CARD-FISH it could be shown that a) the detection rate is poor and further

method optimization is necessary, b) CF319a- and ALT1413-targeted organisms are

associated with Fucus spp..

4. Discussion

The idea of this study was stimulated by the fact that marine ‘Cytophaga’ are capable

of polymer-degradation (Balows et al. 1992). It was shown by this non-directed

isolation approach, that, in addition to ‘Cytophaga’, representatives of the Alpha- and

Gammaproteobacteria inhabit the brown algae Fucus spp.. Most of the isolates belong

to the Gammaproteobacteria, which matches findings of previous studies, in which

the term ‘opportunistic’ bacteria was suggested for the fast-responding

representatives of Alteromonas sp., Pseudoalteromonas sp., and Vibrio sp.. The

isolates of this study were obtained from colonies which were the first to appear on

spread plates. It is therefore not surprising that supposed fast-responding and/or fast-

growing organisms dominate the picture.

Technically, all testing for C-sources was carried out with single C-sources at a time.

However, to bring cells to grow at all, trace amounts of yeast extract (YE) and

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tryptone (T) had to be added to the medium. The sole addition of YE + T to the basic

medium without any other C-source did not result in significant growth. This was

tested for Alteromonas sp. and Pseudoalteromonas sp. (data not shown).

In comparison to agar, fucoidan has not been used for building biomass by most of the

isolates. Cellulophaga sp. strain 5c showed not a slightest trace of growth after 44

hrs. Inspite of being technically a well available compound, it appears to be not as

utilizable as agar. Enzymes for the degradation process might be missing. The fucose,

on the contrary, triggered growth, even though not always as pronounced as galactose

or glucose.

The most efficient - given you consider the doubling time Td as a measure of efficient

substrate utilization – isolate was Pseudoalteromonas sp. with a Td of 0.3 h in Glucose

and 0.7 h and 0.6 h in Galactose and Glucose, respectively. This again, supports other

observations of Pseudoalteromonas spp. being a bacterium with an ‘opportunistic’ life

style. The attempt to check for different substrate and nutrient condition preferences

in North Sea bacterioplankton in dilution enrichments ended in all treatments in

communities dominated by Pseudoalteromonas (unpublished). Cellulophaga spp. on

the contrary might prefer an attached-living lifestyle and is thus in the given

experimental setup not growing under optimal conditions.

5. References

Amann R, Binder BJ, Olson RJ, Chisholm SW, Devereux R, Stahl DA (1990):

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for

analyzing mixed microbial populations. AEM 56 (6) 1919-1925

Balows A, Trüpfer HG, Dwarkin M, Harder W, Schleifer K-H (1992): The Prokaryotes.Springer Verlag, New York

Daims H, Bruhl A, Amann R, Schleifer K-H, Wagner M (1990): The domain-specific

probe EUB338 is insufficient fort he detection of all bacteria: Development and

evaluation of a more comprehensive probe set. Syst. Appl. Microbiol. 22, 434-444

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Descamps V, Colin S, Lahaye M, Jam M, Richard C, Potin P, Barbeyron T, Yvin J-C,

Kloareg B (2006): Isolation and culture of a marine bacterium degrading the sulfated

fucans from marine brown algae. Mar. Biotech. 8, 27-39

Eilers H, Pernthaler J, Gloeckner FO, Amann R (2000): Culturabilty and in situ

abundance of pelagic bacteria from the North Sea. AEM 66 (7) 3044-3051

Manz W, Amann R, Ludwig, W, Vancanneyt M, Schleifer K-H(1996): Application of a

suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of

the phylum cytophaga-flavobacter-bacteroidetes in the natural environment.

Microbiology 142, 1097-1106

Microbial Diversity 2006 course hand out

Pernthaler A, Pernthaler J, Amann, R (2004): Sensitive multi-color fluorescence in situ

hybridization for the identification of environmental microorganisms. In G. Kowalchuk

(ed.), Molecular Microbial Ecology Manual. Kluwer Academic Press, Dordrecht / Boston

/ London.

Wallner G, Amann R (1993): Probing activated sludge with oligonucleotides specific for

proteobacteria: inadequacy of culture-dependent methods for describing microbial

community structure. AEM 59, 1520-1525

Appendix

Tab. Aa.1: General overview - Media used in this study."Agar-Agar"       "Fucoidan/Gel-rite"

SW agar          

Tryptone (Difco)     See SW agar, instead of agar

Yeast extract (Difco)       use 0.3% fucoidan

Agar, washed 1,5 % (or without for liquid)   and 1,0% gelrite

in seawater base        

adjust pH to 7.2        

autoclave          

cool to 60 degree C        

add 50 µg/ml cycloheximide and pour plates    

           

1 x Seawater base        

water 20 l      

NaCl 400 g      

MgCl2*6H2O 60 g      

CaCl2*2H2O 3 g      

KCl 10 g      

           

keep in clean Nalgene platic bottle, not sterile    

C-sources polysaccharides agar or fucoidan 10 mM  

  galactose or fucose or glucose    

buffer 1 M MOPS, pH 7,2 final: 5 mM    

N Ammonium Chloride final: 5 mM, from 100 x stock

P100 x Phosphate Solution, 150 mM, pH7,2

finfal: 1.5 mM, from 150 mMstock

    Potassium Phosphate    

S 1 M Sodium Sulfate final: 0.25 mM  

TE 1000 x HCl-Dissolved Trace Elemts Stock Solution to 1 l add 0.1 ml

Tab Aa.2: The medium used without any C-sources.Medium in 1 x Seawater base  

1 x sea water base 800 ml

Tryptone (T) 0,1 g

Yeast extract (YE) 0,1 g

   

adjust pH to 7.2  

   

1 M MOPS, ph 7,2 5 ml

5 M NH4Cl 1 ml

150 mM KPO4 10 ml

1 M H2SO4 0,25 ml

1000x TE solution 0,1 ml

   filll up to 1 l with 1 x Seawaterbase  

autoclave and cool down to 60°C  

   

1000x Cyclohexamide 1 ml

Tab. Aa.3: Isolates and their closest relatives according to distance matrixanalysis. Species in bold were chosen for further analysis.

# of closest relative

# Habitat Cultiv. nucleotides isolate described isolate

6-12 3 a 1223 Vibrio chagasii 98,66-3a 2 b 602 Vibrio sp. SR2 99,7 6-10 2 a 946

Vibrio alginolyticus 100,06-3b 2 b 1394 6-10 100,0 Vibrio alginolyticus 100,06-1a 1 b 1388 6-10 99,7

Vibrio alginolyticus 99,66-3c 2 b 1393 Vibrio midae 98,76-9 2 a 1374 6-1a 99,6

Vibrio natriegens 99,56-11 2 b 1188 Vibrio sp. HB-8 99,6 Vibrio fortis 99,96-5b 3 b 1352 6-6 100,0

Alteromonas stellaepolaris 99,96-6 3 b 690 6-5b 100,0 Alteromonas stellaepolaris 99,9

6-5a 3 b 1354 Alteromonas sp. R10SW13 99,3Alteromonas macleodii 99,0

6-1b 1 b 1372 Pseudoalteromonas sp. NJ345 99,1 Pseudoalteromonas atlantica 97,96-13 2 b 701 Pseudoalteromonas sp. NJ345 99,1

Pseudoalteromonas atlantica 99,06-8 1 b 1373 Cobetia sp. 37 100,0 Cobetia marina 100,06-7b 1 b 280 6-86-4 2 b 776 Halomonas sp. 100,0 Halomonas elongata 94,8

6-4b 2 b 657 gammaproteobacterium 97,1 close to Cellvibrio 6-7a 1 b 1312 Stappia sp. 100,0 Stappia aggregata 100,0

6-5c 3 a 698 Cellulophaga lytica 99,9

6-2 1 b 1339 Cellulophaga lytica 99,9

1 anaerobically decaying brown algae2 aerobically decaying brown algae3 intact brown algaea anaerobic

Tab. Aa.4: Cell densities of 5 isolates as measured in growth experiments on thepolymers agar and fucoidan.

Agar Fucoidantime[h] Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Cellulo-

alteromonas phaga monas phaga alteromonas phaga monas phaga

0 0,011 0,004 0,045 0,033 -0,027 0,003 0,016 0,015 0,008 0,011

1 0,001 0,005 0,007 0,010 -0,021 0,004 0,016 0,012 0,008 0,011

2 0,006 0,001 0,006 0,010 -0,021 0,007 0,015 0,011 0,009 0,013

3 0,013 -0,001 0,006 0,007 -0,024 0,019 0,015 0,014 0,011 0,011

4 0,071 0,002 0,009 0,010 -0,021 0,078 0,015 0,013 0,008 0,008

5 0,141 0,002 0,013 0,009 -0,021 0,140 0,015 0,013 0,006 0,012

6 0,263 0,002 0,017 0,007 -0,019 0,186 0,016 0,018 0,009 0,013

7 0,332 0,000 0,025 0,006 -0,023 0,181 0,016 0,014 0,008 0,013

8 0,366 -0,001 0,048 0,004 -0,019 0,173 0,015 0,013 0,006 0,012

9 0,375 0,001 0,077 0,006 -0,012 0,167 0,015 0,018 0,008 0,012

10 0,374 0,003 0,104 0,005 -0,011 0,173 0,018 0,019 0,008 0,013

11 0,372 0,012 0,132 0,009 0,002 0,170 0,016 0,022 0,012 0,012

12 0,374 0,020 0,154 0,008 0,009 0,170 0,016 0,029 0,008 0,013

13 0,367 0,037 0,180 0,011 0,018 0,163 0,014 0,022 0,009 0,011

14 0,360 0,049 0,201 0,014 0,027 0,163 0,012 0,028 0,008 0,013

15 x 0,077 0,255 0,043 0,043 x 0,018 0,036 0,007 0,013

16 x 0,109 0,312 0,071 0,058 x 0,017 0,039 0,007 0,013

17 x 0,135 0,350 0,118 0,067 x 0,016 0,044 0,007 0,013

18 x 0,154 0,377 0,215 0,075 x 0,013 0,058 0,010 0,012

19 x 0,176 0,405 0,351 0,089 x 0,018 0,051 0,009 0,016

20 x 0,192 0,421 0,417 0,092 x 0,016 0,053 0,009 0,014

21 x 0,210 0,426 0,435 0,103 x 0,017 0,058 0,009 0,012

30 x 0,246 0,455 0,462 0,108 x 0,017 0,046 0,029 0,009

44 x x x x x 0,119 0,024 0,065 0,025 0,009

51 x x x x x x 0,033 0,064 x x

Tab. Aa.5: Cell densities of 5 isolates as measured in growth experiments on the sugar galactose, fucose, and glucose.Galactose Fucose Glucose

time[h] Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Cellulo- Pseudo- Cellulo- Cellvibrio' Altero- Cellulo-

alteromonas phaga monas phaga alteromonas phaga monas phaga alteromonas phaga monas phaga

0,0 0,010 0,006 0,006 0,003 0,003 0,001 0,001 0,001 0,005 -0,001 -0,003 -0,002 0,006 0,001 -0,003

1,3 0,013 0,007 0,005 0,009 0,002 0,010 0,005 0,005 0,018 0,006 0,003 0,001 0,009 0,006 -0,001

2,2 0,054 0,010 0,006 0,018 0,009 0,035 0,001 0,002 0,012 -0,002 0,047 0,009 0,020 0,025 0,009

2,7 0,110 0,010 0,006 0,031 0,006 0,076 0,001 0,003 0,022 -0,001 0,106 0,009 0,019 0,039 0,007

3,2 0,185 0,009 0,010 0,052 0,003 0,112 0,002 0,007 0,031 0,000 0,158 0,007 0,024 0,058 0,006

3,7 0,258 0,007 0,017 0,066 0,006 0,146 0,006 0,011 0,042 0,002 0,199 0,010 0,035 0,078 0,005

4,4 0,355 0,010 0,023 0,112 0,003 0,158 0,008 0,017 0,075 0,006 0,242 0,011 0,057 0,126 0,006

5,1 0,444 0,010 0,045 0,185 0,003 0,171 0,008 0,035 0,101 0,005 0,259 0,009 0,101 0,219 0,004

5,8 0,496 0,015 0,068 0,294 0,007 0,164 0,004 0,043 0,135 0,004 0,290 0,009 0,158 0,315 0,009

6,8 0,558 0,013 0,103 0,433 0,003 0,165 0,006 0,066 0,158 0,004 0,323 0,007 0,320 0,474 0,008

7,8 0,599 0,013 0,184 0,519 0,008 0,165 0,007 0,086 0,164 0,008 0,304 0,011 0,428 0,580 0,006

11,8 0,709 0,017 0,516 0,825 0,008 0,168 0,012 0,116 0,178 0,014 0,425 0,017 0,554 0,814 0,018

13,0 0,741 0,018 0,570 0,893 0,010 0,157 0,015 0,126 0,179 0,017 0,429 0,028 0,597 0,887 0,023

14,2 0,750 0,032 0,613 0,953 0,014 0,155 0,025 0,134 0,181 0,025 0,414 0,033 0,611 0,936 0,037

15,3 0,753 0,048 0,634 1,004 0,026 0,160 0,034 0,139 0,180 0,045 0,442 0,057 0,623 0,995 0,057

33,3 0,518 0,076 0,546 1,128 0,046 0,130 0,103 0,147 0,178 0,129 0,615 0,151 0,593 0,852 0,113

Tab. Aa.6: Doubling times Td of all 5 isolates. Isolate C-source Td [h]

Alteromonas sp. agar 1,4

Galactose 0,9

Fucose 1,0

Glucose 1,0

Pseudoalteromonas sp. Agar 0,9

Fucoidan 0,8

Galactose 0,7

Fucose 0,6

Glucose 0,3

Cellvibrio-like Agar 1,8

Fucoidan 3,9

Galactose 0,9

Fucose 0,7

Glucose 1,1

Cellulophaga sp. 5c Agar 1,1

Fucoidan b.d.

Galactose b.d.

Fucose b.d.

Glucose b.d.

Cellulophaga sp. 2 Agar 1,6

2 Fucoidan b.d.

Galactose b.d.

Fucose b.d.

  Glucose b.d.

b.d. below detection

Cellulophaga (2)

0,00

0,04

0,08

0,12

0,16

6 8 10 12 14 16

time [h]

OD

600

Galactose

Fucose

Glucose

Pseudoalteromonas

-0,10

0,10

0,30

0,50

0,70

0,90

0 2 4 6 8 10 12 14

time [h]

OD

600

Galactose

Fucose

Glucose

Alteromonas

0,00

0,20

0,40

0,60

0,80

1,00

1,20

0 2 4 6 8 10 12 14 16

time [h]

OD

600

Galactose

Fucose

Glucose

'Cellvibrio relative'

0,00

0,20

0,40

0,60

0,80

2 4 6 8 10 12 14

time [h]

OD

600

Galactose

Fucose

Glucose

Cellulophaga (5c)

-0,02

0,00

0,02

0,04

0,06

0,08

0,10

0,12

0,14

0 2 4 6 8 10 12 14 16

time [h]

OD

600

Galactose

Fucose

Glucose

Fig. Ab.3: Growth curvesof the 5 isoaltes growingon either galactose, fucoseor glucose.

Cellulophaga (2)

-0,100

0,000

0,100

0,200

0,300

8 10 12 14 16 18 20 22 24

]time [h

OD

600

Agar

Fucoidan

'Cellvibrio relative'

0,000

0,100

0,200

0,300

0,400

0,500

4 6 8 10 12 14 16 18 20 22

]time [h

OD

600

Agar

Fucoidan

Pseudoalteromonas

0,000

0,100

0,200

0,300

0,400

0 5 10 15

]time [h

OD

600

Agar

Fucoidan

Alteromonas

0,000

0,100

0,200

0,300

0,400

0,500

8 13 18 23 28 33

] time [h

OD

600

Agar

Fucoidan

Cellulophaga (5c)

-0,100

0,000

0,100

0,200

8 13 18

]time [h

OD

600

Agar

Fucoidan

Fig. Ab.2: Growth curves of 5 isolates growing on the polymers agar or fucoidan,respectively.

ll

Fig. Ab.1: The isolates which werechaosen for further analysis.A Alteromonas sp., B Pseudoalteromonassp. C Cellvibrio-like, D + E Cellulophagaspp.

A

B C DE

E

Appendix C

Protocols

I would like to thank the faculty and the TAs for making this course the great

experience it was. Especially, Jean and Kou-San were more than patient whenever

I chose the complicated way to think about the simplest things.

Thank you Team 2 – you are special, you are different, and you put up with me!

Last but not least, thank you to the whole class Microbial Diversity 2006. It was a

challenging and rewarding experience meeting all of you!

I appreciate the funding through the Gordon and Betty Moore Foundation and the

Daniel and Edith Grosch Fund. Without them I would not have had this special

summer in Woods Hole.