Polymer-degrading bacteria associated with the
brown algae Fucus spp.
Elke Allers
Carried out as an Individual Project within the
Microbial Diversity Course 2006
at the Marine Biological Laboratory in Woods Hole
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.
-1-
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
-2-
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 PCRmix 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
-3-
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 fastgrowing 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
-4-
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
-5-
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
-6-
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
and 1,0% gelrite
Agar, washed
1,5 % (or without for liquid)
in seawater base
adjust pH to 7.2
autoclave
add 50 µg/ml cycloheximide and pour plates
cool to 60 degree C
water
20 l
NaCl
400 g
MgCl2*6H2O
60 g
CaCl2*2H2O
3 g
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
P
100 x Phosphate Solution, 150 mM, pH
7,2
finfal: 1.5 mM, from 150 mM
stock
S
1 M Sodium Sulfate
TE
1000 x HCl-Dissolved Trace Elemts Stock Solution
1 x Seawater base
KCl
Potassium Phosphate
final: 0.25 mM
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
1 M H2SO4
1000x TE solution
10 ml
0,25 ml
0,1 ml
filll up to 1 l with 1 x Seawater
base
autoclave and cool down to 60°C
1000x Cyclohexamide
1 ml
Tab. Aa.3: Isolates and their closest relatives according to distance matrix
analysis. Species in bold were chosen for further analysis.
# of
#
closest relative
Habitat Cultiv. nucleotides
isolate
described isolate
6-12
6-3a
6-10
3 a
2 b
2 a
1223
602
946
Vibrio sp. SR2
Vibrio chagasii
6-3b
2 b
1394
6-10
100,0
6-1a
1 b
1388
6-10
99,7
6-3c
6-9
2 b
2 a
1393
1374
6-1a
99,6
6-11
2 b
1188
Vibrio sp. HB-8
99,6
6-5b
3 b
1352
6-6
100,0
6-6
3 b
690
6-5b
100,0
6-5a
3 b
1354
Alteromonas sp. R10SW13
99,3
6-1b
1 b
1372
Pseudoalteromonas sp. NJ345
99,1
6-13
2 b
701
Pseudoalteromonas sp. NJ345
99,1
6-8
1 b
1373
Cobetia sp. 37
Vibrio alginolyticus
100,0
Vibrio alginolyticus
100,0
Vibrio alginolyticus
Vibrio midae
99,6
98,7
Vibrio natriegens
99,5
Vibrio fortis
99,9
Alteromonas stellaepolaris
99,9
Alteromonas stellaepolaris
99,9
Alteromonas macleodii
99,0
Pseudoalteromonas atlantica
97,9
Pseudoalteromonas atlantica
99,0
100,0
Cobetia marina
6-7b
6-4
1 b
2 b
6-4b
6-7a
2 b
1 b
280
776
657
1312
6-8
Halomonas sp.
gammaproteobacterium
Stappia sp.
98,6
99,7
100,0
100,0
Halomonas elongata
94,8
97,1 close to Cellvibrio
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
2
3
a
anaerobically decaying brown algae
aerobically decaying brown algae
intact brown algae
anaerobic
Tab. Aa.4: Cell densities of 5 isolates as measured in growth experiments on the
polymers agar and fucoidan.
Agar
Fucoidan
time
[h] PseudoCellulo- Cellvibrio' Altero- Cellulo- PseudoCellulo- Cellvibrio' Altero- Celluloalteromonas phaga
monas phaga alteromonas phaga
monas phaga
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
30
44
51
0,011
0,001
0,006
0,013
0,071
0,141
0,263
0,332
0,366
0,375
0,374
0,372
0,374
0,367
0,360
x
x
x
x
x
x
x
x
x
x
0,004
0,005
0,001
-0,001
0,002
0,002
0,002
0,000
-0,001
0,001
0,003
0,012
0,020
0,037
0,049
0,077
0,109
0,135
0,154
0,176
0,192
0,210
0,246
x
x
0,045
0,007
0,006
0,006
0,009
0,013
0,017
0,025
0,048
0,077
0,104
0,132
0,154
0,180
0,201
0,255
0,312
0,350
0,377
0,405
0,421
0,426
0,455
x
x
0,033
0,010
0,010
0,007
0,010
0,009
0,007
0,006
0,004
0,006
0,005
0,009
0,008
0,011
0,014
0,043
0,071
0,118
0,215
0,351
0,417
0,435
0,462
x
x
-0,027
-0,021
-0,021
-0,024
-0,021
-0,021
-0,019
-0,023
-0,019
-0,012
-0,011
0,002
0,009
0,018
0,027
0,043
0,058
0,067
0,075
0,089
0,092
0,103
0,108
x
x
0,003
0,004
0,007
0,019
0,078
0,140
0,186
0,181
0,173
0,167
0,173
0,170
0,170
0,163
0,163
x
x
x
x
x
x
x
x
0,119
x
0,016
0,016
0,015
0,015
0,015
0,015
0,016
0,016
0,015
0,015
0,018
0,016
0,016
0,014
0,012
0,018
0,017
0,016
0,013
0,018
0,016
0,017
0,017
0,024
0,033
0,015
0,012
0,011
0,014
0,013
0,013
0,018
0,014
0,013
0,018
0,019
0,022
0,029
0,022
0,028
0,036
0,039
0,044
0,058
0,051
0,053
0,058
0,046
0,065
0,064
0,008
0,008
0,009
0,011
0,008
0,006
0,009
0,008
0,006
0,008
0,008
0,012
0,008
0,009
0,008
0,007
0,007
0,007
0,010
0,009
0,009
0,009
0,029
0,025
x
0,011
0,011
0,013
0,011
0,008
0,012
0,013
0,013
0,012
0,012
0,013
0,012
0,013
0,011
0,013
0,013
0,013
0,013
0,012
0,016
0,014
0,012
0,009
0,009
x
Tab. Aa.5: Cell densities of 5 isolates as measured in growth experiments on the sugar galactose, fucose, and glucose.
Galactose
time
[h]
0,0
1,3
2,2
2,7
3,2
3,7
4,4
5,1
5,8
6,8
7,8
11,8
13,0
14,2
15,3
33,3
Fucose
Glucose
PseudoCellulo- Cellvibrio' Altero- CelluloPseudoCellulo- Cellvibrio' Altero- CelluloPseudoCellulo- Cellvibrio' Altero- Celluloalteromonas phaga
monas phaga alteromonas phaga
monas phaga alteromonas phaga
monas phaga
0,010
0,013
0,054
0,110
0,185
0,258
0,355
0,444
0,496
0,558
0,599
0,709
0,741
0,750
0,753
0,518
0,006
0,007
0,010
0,010
0,009
0,007
0,010
0,010
0,015
0,013
0,013
0,017
0,018
0,032
0,048
0,076
0,006
0,005
0,006
0,006
0,010
0,017
0,023
0,045
0,068
0,103
0,184
0,516
0,570
0,613
0,634
0,546
0,003
0,009
0,018
0,031
0,052
0,066
0,112
0,185
0,294
0,433
0,519
0,825
0,893
0,953
1,004
1,128
0,003
0,002
0,009
0,006
0,003
0,006
0,003
0,003
0,007
0,003
0,008
0,008
0,010
0,014
0,026
0,046
0,001
0,010
0,035
0,076
0,112
0,146
0,158
0,171
0,164
0,165
0,165
0,168
0,157
0,155
0,160
0,130
0,001
0,005
0,001
0,001
0,002
0,006
0,008
0,008
0,004
0,006
0,007
0,012
0,015
0,025
0,034
0,103
0,001
0,005
0,002
0,003
0,007
0,011
0,017
0,035
0,043
0,066
0,086
0,116
0,126
0,134
0,139
0,147
0,005
0,018
0,012
0,022
0,031
0,042
0,075
0,101
0,135
0,158
0,164
0,178
0,179
0,181
0,180
0,178
-0,001
0,006
-0,002
-0,001
0,000
0,002
0,006
0,005
0,004
0,004
0,008
0,014
0,017
0,025
0,045
0,129
-0,003
0,003
0,047
0,106
0,158
0,199
0,242
0,259
0,290
0,323
0,304
0,425
0,429
0,414
0,442
0,615
-0,002
0,001
0,009
0,009
0,007
0,010
0,011
0,009
0,009
0,007
0,011
0,017
0,028
0,033
0,057
0,151
0,006
0,009
0,020
0,019
0,024
0,035
0,057
0,101
0,158
0,320
0,428
0,554
0,597
0,611
0,623
0,593
0,001
0,006
0,025
0,039
0,058
0,078
0,126
0,219
0,315
0,474
0,580
0,814
0,887
0,936
0,995
0,852
-0,003
-0,001
0,009
0,007
0,006
0,005
0,006
0,004
0,009
0,008
0,006
0,018
0,023
0,037
0,057
0,113
Tab. Aa.6: Doubling times Td of all 5 isolates.
Isolate
Alteromonas sp.
Pseudoalteromonas sp.
Cellvibrio-like
Cellulophaga sp. 5c
Cellulophaga sp. 2
C-source
agar
1,4
Galactose
0,9
Fucose
1,0
Glucose
1,0
Agar
0,9
Fucoidan
0,8
Galactose
0,7
Fucose
0,6
Glucose
0,3
Agar
1,8
Fucoidan
3,9
Galactose
0,9
Fucose
0,7
Glucose
1,1
Agar
1,1
Fucoidan
b.d.
Galactose
b.d.
Fucose
b.d.
Glucose
b.d.
Agar
2 Fucoidan
b.d. below detection
Td [h]
1,6
b.d.
Galactose
b.d.
Fucose
b.d.
Glucose
b.d.
1,20
0,90
Galactose
Pseudoalteromonas
Galactose
1,00
Fucose
0,70
OD 600
OD 600
Glucose
0,80
Glucose
0,50
Alteromonas
Fucose
0,60
0,40
0,30
0,20
0,10
0,00
-0,10 0
2
4
6
8
10
12
14
0
2
4
6
8
time [h]
10
12
14
16
time [h]
0,80
'Cellvibrio relative'
Galactose
0,60
OD 600
Fig. Ab.3: Growth curves
of the 5 isoaltes growing
on either galactose, fucose
or glucose.
Fucose
Glucose
0,40
0,20
0,00
2
4
6
8
10
12
14
time [h]
0,16
Galactose
0,14
Cellulophaga (2)
Fucose
0,12
Fucose
Glucose
0,10
Glucose
0,08
0,08
OD 600
OD 600
Cellulophaga (5c)
Galactose
0,12
0,06
0,04
0,04
0,02
0,00
0,00
6
8
10
12
time [h]
14
16
-0,02
0
2
4
6
8
time [h]
10
12
14
16
Fig. Ab.2: Growth curves of 5 isolates growing on the polymers agar or fucoidan,
respectively.
0,400
Agar
Fucoidan
0,400
OD600
0,300
OD600
0,500
Agar
Fucoidan
0,200
0,100
0,300
Alteromonas
0,200
0,100
Pseudoalteromonas
0,000
0
5
10
15
0,000
8
]time [h
13
18
23
28
33
] time [h
0,500
Agar
Fucoidan
0,400
0,300
'Cellvibrio relative'
0,200
0,300
OD600
OD600
Cellulophaga (2)
Agar
Fucoidan
0,200
0,100
0,100
0,000
8
10
12
14
16
0,000
4
6
8
10
12
14
16
18
0,200
Cellulophaga (5c)
OD600
0,100
0,000
8
13
-0,100
]time [h
22
-0,100
]time [h
]time [h
Agar
Fucoidan
20
18
18
20
22
24
A
Fig. Ab.1: The isolates which were
chaosen for further analysis.
A Alteromonas sp., B Pseudoalteromonas
sp. C Cellvibrio-like, D + E Cellulophaga
spp.
B
C
ll
D
E
E
Appendix C
Protocols
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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.
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