Heterotrophic Bacteria Associated with
Cyanobacteria in Recreational and Drinking Water
Katri Berg
Department of Applied Chemistry and Microbiology
Division of Microbiology
Faculty of Agriculture and Forestry
University of Helsinki
Academic Dissertation in Microbiology
To be presented, with the permission of the Faculty of Agriculture and Forestry
of the University of Helsinki, for public criticism in Walter hall (2089) at
Agnes Sjöbergin katu 2, EE building on December 11th at 12 o’clock noon.
Helsinki 2009
Supervisors:
Docent Jarkko Rapala
National Supervisory Authority for Welfare and Health, Finland
Docent Christina Lyra
Department of Applied Chemistry and Microbiology
University of Helsinki, Finland
Academy Professor Kaarina Sivonen
Department of Applied Chemistry and Microbiology
University of Helsinki, Finland
Reviewers:
Professor Marja-Liisa Hänninen
Department of Food and Environmental Hygiene
University of Helsinki, Finland
Docent Stefan Bertilsson
Department of Ecology and Evolution
Uppsala University, Sweden
Opponent:
Professor Agneta Andersson
Department of Ecology and Environmental Sciences
Umeå University, Sweden
Printed Yliopistopaino, Helsinki, 2009
Layout Timo Päivärinta
ISSN 1795-7079
ISBN 978-952-10-5892-9 (paperback)
ISBN 978-952-10-5893-6 (PDF)
e-mail [email protected]
Front cover picture
DAPI stained cells of Anabaena cyanobacterium and Paucibacter toxinivorans strain
2C20.
CONTENTS
LIST OF ORIGINAL PAPERS
THE AUTHOR’S CONTRIBUTION
ABBREVIATIONS
ABSTRACT
TIIVISTELMÄ (ABSTRACT IN FINNISH)
1 INTRODUCTION ..................................................................................................... 1
1.1 Cyanobacteria ........................................................................................................ 1
1.2 Cyanobacterial mass occurrences ......................................................................... 1
1.2.1 Bloom forming cyanobacteria ..................................................................... 1
1.3 Cyanobacterial toxins ............................................................................................ 2
1.3.1 Hepatotoxins ................................................................................................ 2
1.3.2 Neurotoxins ................................................................................................. 3
1.3.3 Other toxins of cyanobacteria ...................................................................... 4
1.3.4 Toxins analyses ............................................................................................ 5
1.3.5 Persistence of cyanobacterial toxins ............................................................ 6
1.4 Aquatic cyanobacteria and heterotrophic bacteria ................................................ 6
1.4.1 Aquatic bacteria ........................................................................................... 7
1.4.2 Interactions between heterotrophic bacteria and cyanobacteria .................. 7
1.5 Bacterial characterisation ...................................................................................... 8
1.5.1 Isolation of bacteria ..................................................................................... 9
1.5.2 Phenotypic analyses .................................................................................... 9
1.5.3 Molecular analyses in taxonomic identification of bacteria ...................... 10
1.5.4 Genetic analyses in assessment of physiological features ......................... 11
1.5.5 Statistical analyses of multivariate data .................................................... 11
1.6 Problems associated with cyanobacterial water blooms ..................................... 12
1.6.1 Adverse human health effects associated with
cyanobacterial water blooms ..................................................................... 12
1.6.2 Cyanobacterial water blooms and drinking water ..................................... 14
1.6.3 Cyanobacterial toxins and the drinking water treatment ........................... 14
1.6.4 Combined effects of cyanobacterial water blooms and
heterotrophic bacteria on the drinking water quality ................................. 15
2 AIMS OF THE STUDY .......................................................................................... 18
3 MATERIALS AND METHODS ............................................................................ 19
3.1 Water samples and isolated bacterial strains ....................................................... 19
3.2 Methods................................................................................................................ 19
4 RESULTS ................................................................................................................. 21
4.1 Heterotrophic bacteria associated with cyanobacteria ........................................ 21
4.1.1 Taxonomic assignment of the isolated bacterial strains ............................. 21
4.1.2 Bacteria degrading cyanobacterial hepatotoxins:
Paucibacter toxinivorans gen. nov. sp. nov. .............................................. 21
4.1.3 Virulence potential of the heterotrophic bacteria associated
with cyanobacteria ..................................................................................... 24
4.1.4 Effects of the heterotrophic bacteria on the growth of cyanobacteria ........ 25
4.2 Selectivity of the used growth media .................................................................. 26
4.3 Drinking water treatment efficiency ................................................................... 28
4.3.1 Removal of toxins by the water treatment .................................................. 28
4.3.2 Removal of planktonic cells by the water treatment................................... 29
4.3.3 Taxonomy of the heterotrophic bacteria isolated from drinking water....... 30
5 DISCUSSION .......................................................................................................... 31
5.1 Characteristics of the isolated heterotrophic bacteria .......................................... 31
5.2 Drinking water treatment efficiency .................................................................... 34
6 CONCLUSIONS ...................................................................................................... 36
7 ACKNOWLEDGEMENTS..................................................................................... 38
8 REFERENCES......................................................................................................... 39
APPENDIX 1 The bacterial strains analysed in this study, water type of their
origin, isolation medium used, length of the acquired 16S rRNA gene sequence
and its accession number, and the taxonomic assignment of the strains ..................... 52
LIST OF ORIGINAL PAPERS
I
K. A. Berg, C. Lyra, K. Sivonen, L. Paulin, S. Suomalainen, P. Tuomi, and J.
Rapala. 2009. High diversity of cultivable heterotrophic bacteria in association
with cyanobacterial water blooms. ISME J. 3: 314–325.
II
K. A. Berg, C. Lyra, R. M. Niemi, B. Heens, K. Hoppu, K. Erkomaa, K. Sivonen,
and J. Rapala. Virulence genes in Aeromonas strains isolated from cyanobacterial
water bloom samples associated with human health symptoms. Manuscript,
submitted to ISME J.
III J. Rapala, K. A. Berg, C. Lyra, R. M. Niemi, W. Manz, S. Suomalainen, L. Paulin,
and K. Lahti. 2005. Paucibacter toxinivorans gen. nov., sp. nov., a bacterium that
degrades cyclic cyanobacterial hepatotoxins microcystins and nodularin. Int. J.
Syst. Evol. Microbiol. 55: 1563–1568.
IV J. Rapala, M. Niemelä, K. A. Berg, L. Lepistö, and K. Lahti. 2006. Removal of
cyanobacteria, cyanotoxins, heterotrophic bacteria and endotoxins at an operating
surface water treatment plant. Wat. Sci. Technol. 54(3): 23–28.
The original papers were reproduced with the kind permission of the copyright holders.
THE AUTHOR’S CONTRIBUTION
I
Katri Berg participated in designing of the experiment, isolated the majority of the
bacterial strains and performed their taxonomic identification. She interpreted the
results and wrote the paper.
II
Katri Berg participated in designing of the experiment and isolation of the bacterial
strains. She also performed taxonomic identification of the bacteria and the
statistical analyses of the data, interpreted the results and wrote the paper.
III Katri Berg participated in designing of the experiment, studied the presence of
flagellae and bacteriochlorophyll of the strains, performed the toxin degradation
tests and interpreted the results of the toxin analyses. She wrote a part of the
paper.
IV Katri Berg participated in the isolation of the bacterial strains, the toxin analysis
and the taxonomic identification of the strains.
ABBREVIATIONS
Adda
AH
BA
BLAST
CAP
CFU
DISTLMf
ELISA
EU
GLC
HPLC
LD50
Microcystin-LR
Microcystin-YR
NCBI
PAUP
PCO
PCR
PHYLIP
R2A
SAA
TSA
UPGMA
WHO
(2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10phenyldeca-4,6-dienoic acid
Aeromonas hydrophila
Blood agar
Basic local alignment search tool
Program for canonical analysis of principal coordinates
Colony forming unit
Program for distance-based multivariate analysis for a linear
model using forward selection
Enzyme-linked immunosorbent assay
Endotoxin unit
Gas-liquid chromatography
High-performance liquid chromatography
Lethal dose, 50%
Microcystin with leusine as aminoacid 2 and arginine as amino
acid 4
Microcystin with tyrosine as aminoacid 2 and arginine as amino
acid 4
National Center for Biotechnology Information
Phylogenetic analysis using parsimony
Program for principal coordinate analysis
Polymerase chain reaction
Phylogeny inference package
Reasoner’s 2A medium
Starch ampicillin agar
Trypticase soy agar
Unweighted pair group method with arithmetic averages
World Health Organization
ABSTRACT
Cyanobacterial mass occurrences, also known as water blooms, have been associated
with adverse health effects of both humans and animals. They can also be a burden
to drinking water treatment facilities. Risk assessments of the blooms have generally
focused on the cyanobacteria themselves and their toxins. However, heterotrophic
bacteria thriving among cyanobacteria may also be responsible for many of the adverse
health effects, but their role as the etiological agents of these health problems is poorly
known. In addition, studies on the water purification efficiency of operating water
treatment plants during cyanobacterial mass occurrences in their water sources are rare.
In the present study, over 600 heterotrophic bacterial strains were isolated from
natural freshwater, brackish water or from treated drinking water. The sampling sites
were selected as having frequent cyanobacterial occurrences in the water bodies or in
the water sources of the drinking water treatment plants. In addition, samples were taken
from sites where cyanobacterial water blooms were surmised to have caused human
health problems. The isolated strains represented bacteria from 57 different genera of the
Gamma-, Alpha- or Betaproteobacteria, Actinobacteria, Flavobacteria, Sphingobacteria,
Bacilli and Deinococci classes, based on their partial 16S rRNA sequences. Several
isolates had no close relatives among previously isolated bacteria or cloned 16S rRNA
genes of uncultivated bacteria. The results show that water blooms are associated with a
diverse community of cultivable heterotrophic bacteria.
Chosen subsets of the isolated strains were analysed for features such as their
virulence gene content and possible effect on cyanobacterial growth. Of the putatively
pathogenic haemolytic strains isolated in the study, the majority represented the genus
Aeromonas. Therefore, the Aeromonas spp. strains isolated from water samples associated
with adverse health effects were screened for the virulence gene types encoding for
enterotoxins (ast, alt and act/aerA/hlyA), flagellin subunits (flaA/flaB), lipase (lip/pla/
lipH3/alp-1) and elastase (ahyB) by PCR. The majority (90%) of the Aeromonas strains
included one or more of the six screened Aeromonas virulence gene types. The most
common gene type was act, which was present in 77% of the strains. The fla, ahyB and
lip genes were present in 30–37% of the strains. The prevalence of the virulence genes
implies that the Aeromonas may be a factor in some of the cyanobacterial associated
health problems.
Of the 183 isolated bacterial strains that were studied for possible effects on
cyanobacterial growth, the majority (60%) either enhanced or inhibited growth of
cyanobacteria. In most cases, they enhanced the growth, which implies mutualistic
interactions. The results indicate that the heterotrophic bacteria have a role in the rise
and fall of the cyanobacterial water blooms.
The genetic and phenotypic characteristics and the ability to degrade cyanobacterial
hepatotoxins of 13 previously isolated Betaproteobacteria strains, were also studied. The
strains originated from Finnish lakes with frequent cyanobacterial occurrence. Tested
strains degraded microcystins -LR and -YR and nodularin. The strains could not be
assigned to any described bacterial genus or species based on their genetic or phenotypic
features. On the basis of their characteristics a new genus and species Paucibacter
toxinivorans was proposed for them.
The water purification efficiency of the drinking water treatment processes during
cyanobacterial water bloom in water source was assessed at an operating surface water
treatment plant. Large phytoplankton, cyanobacterial hepatotoxins, endotoxins and
cultivable heterotrophic bacteria were efficiently reduced to low concentrations, often
below the detection limits. In contrast, small planktonic cells, including also possible
bacterial cells, regularly passed though the water treatment. The passing cells may
contribute to biofilm formation within the water distribution system, and therefore lower
the obtained drinking water quality.
The bacterial strains of this study offer a rich source of isolated strains for examining
interactions between cyanobacteria and the heterotrophic bacteria associated with them.
The degraders of cyanobacterial hepatotoxins could perhaps be utilized to assist the
removal of the hepatotoxins during water treatment, whereas inhibitors of cyanobacterial
growth might be useful in controlling cyanobacterial water blooms. The putative
pathogenicity of the strains suggests that the health risk assessment of the cyanobacterial
blooms should also cover the heterotrophic bacteria.
TIIVISTELMÄ (ABSTRACT IN FINNISH)
Syanobakteerien massaesiintymät, sinileväkukinnat, on yhdistetty monenlaisiin
haitallisiin terveysvaikutuksiin, kuten pahoinvointiin, kuumeeseen, päänsärkyyn ja ihosekä vatsaoireisiin. Kukinnat voivat myös aiheuttaa ylimääräistä kuormitusta juomaveden
puhdistuslaitoksille ja heikentää tuotetun juomaveden laatua. Syanobakteerikukintojen
aiheuttamien terveysriskien arviointi on yleensä keskittynyt itse syanobakteereihin ja
niiden tuottamiin toksiineihin vaikka oireiden mahdollisina aiheuttajina voivat toimia
myös kukintojen yhteydessä elävät heterotrofiset bakteerit. Niiden osuus kukintojen
aiheuttamien oireiden synnyssä on kuitenkin pitkälti tuntematon. Myös tutkimukset
juomaveden puhdistuslaitosten vedenpuhdistuksen tehokkuudesta raakavedessä
esiintyvien kukintojen aikana ovat harvinaisia.
Tässä työssä eristettiin yli 600 heterotrofista bakteerikantaa makea- ja murtovetisistä
luonnonvesistöistä sekä puhdistetusta juomavedestä. Vesinäytteiden keräyskohteet oli
valittu vesistöissä tai juomaveden raakavedessä usein toistuvien syanobakteeriesiintymien
perusteella. Näytteitä otettiin myös vesiympäristöistä, joissa syanobakteerikukintojen
epäiltiin aiheuttaneen terveysoireita ihmisille. Eristetyt bakteerikannat edustivat 16S
rRNA -geenisekvenssiensä perusteella 57 eri bakteerisukua jotka kuuluivat Gamma-,
Alpha- ja Betaproteobacteria, Actinobacteria, Flavobacteria, Sphingobacteria,
Bacilli ja Deinococci -luokkiin. Useilla kannoilla ei ollut läheisiä sukulaisia aiemmin
eristettyjen bakteerien tai viljelystä riippumattomilla menetelmillä kerättyjen 16S rRNA
-geenisekvenssien joukossa. Tulosten perusteella syanobakteerikukintojen yhteydessä
esiintyy monipuolinen viljeltävissä olevien heterotrofisten bakteerien yhteisö.
Eristetyistä heterotrofisista bakteerikannoista analysoitiin ominaisuuksia kuten
virulenssigeenien esiintymistä ja mahdollisia vaikutuksia syanobakteerien kasvuun.
Tutkituista hemolyyttisistä ja siten mahdollisesti virulenteista kannoista valtaosa edusti
Aeromonas-sukua. Tulosten perusteella terveysoireisiin liittyneistä vesinäytteistä
eristetyistä Aeromonas-kannoista etsittiin kuutta Aeromonas virulenssigeenityyppiä PCR
menetelmällä. Nämä geenityypit koodaavat enterotoksiineja (ast, alt ja act/aerA/hlyA
geenit), flagelliini-alayksikköjä (flaA/falB), lipaasia (lip/pla/lipH3/alp-1) ja elastaasia
(ahyB). Valtaosa (90 %) tutkituista Aeromonas-kannoista sisälsi yhden tai useamman
geenityypeistä. Niistä yleisin oli act, joka havaittiin 77 % kannoista. Myös fla, ahyB ja
lip -geenejä havaittiin 30–37%:ssa kannoista. Virulenssigeenien yleisyys viittaa siihen,
että Aeromonas-bakteerit saattavat olla yksi syanobakteerikukintoihin yhdistettyjen
oireiden aiheuttajista.
Niistä yli 180 eristetystä bakteerikannasta, joiden kykyä vaikuttaa syanobakteerien
kasvuun tutkittiin, valtaosa (60 %) joko kiihdytti tai esti syanobakteerien kasvua.
Useimmissa tapauksissa vaikutus oli kasvua kiihdyttävä, mikä viittaa mutualistiseen
vuorovaikutukseen. Tulosten perusteella heterotrofiset bakteerit saattavat vaikuttaa
syanobakteerien massaesiintymien muodostumiseen ja kestoon.
Työssä tutkittiin myös kolmentoista aiemmin eristetyn Betaproteobacteria-kannan
geneettisiä ja fenotyyppisiä ominaisuuksia, kuten 16S rRNA geenisekvenssiä ja
entsyymiaktiivisuuksia, ja kykyä hajottaa syanobakteerien tuottamia maksatoksiineita,
mikrokystiineitä ja nodulariinia. Kannat eivät geneettisten ja fenotyyppisten piirteidensä
perusteella kuuluneet aiemmin kuvattuihin bakteerisukuihin tai -lajeihin. Kaikki
tutkitut kannat hajottivat maksatoksiineita. Kantojen geneettisten- ja fenotyyppisiin
ominaisuuksiin perustuen kuvattiin uusi bakteerisuku ja -laji, Paucibacter toxinivorans.
Lisäksi työssä arvioitiin toiminnassa olevan juomavedenveden puhdistuslaitoksen
vedenpuhdistustehokkuutta syanobakteerikukinnan aikana. Suurten kasviplanktonsolujen,
syanobakteerien maksatoksiinien, endotoksiinien ja viljeltävien heterotrofisten bakteerien
määrät vähenivät vedenkäsittelyn aikana tehokkaasti. Sen sijaan pienet planktonsolut
läpäisivät vedenkäsittelyketjun säännöllisesti. Puhdistusketjun läpäisevät solut voivat
edistää biofilmien muodostumista vedenjakeluverkostossa ja siten heikentää käsitellyn
veden laatua.
Työssä käsitellyt heterotrofiset bakteerikannat tarjoavat monipuolisen kokoelman
bakteerikantoja syanobakteerien ja niiden yhteydessä esiintyvien heterotrofisten
bakteerien välisten vuorovaikutusten tutkimiseksi. Syanobakteerien tuottamia
maksatoksiineita hajottavia kantoja voitaisiin mahdollisesti hyödyntää maksatoksiinien
poistamisessa vedenpuhdistuksessa ja syanobakteerien kasvua estäviä kantoja
kukintojen kontrolloinnissa. Kantojen mahdollinen patogeenisyys viittaa siihen, että
myös kukintojen yhteydessä esiintyvät heterotrofiset bakteerit pitäisi ottaa huomioon
syanobakteerikukintoihin liittyvien terveysriskien arvioinnissa.
Introduction
1. INTRODUCTION
1.1 Cyanobacteria
Cyanobacteria are fascinating organisms
that posses features familiar to both
prokaryotic and eukaryotic branches
of the tree of life. Their cell structure
and composition is similar to that of a
prokaryotic cell in that they lack the cell
nucleus and distinctive organelles of
eukaryotic cells. However, in contrast
to typical prokaryotes they are able
to perform oxygenic photosynthesis
(Castenholtz 2001). Evidence of fossilised
cyanobacteria-like organisms date back for
more than 2500 million years and that of
versatile cyanobacterial biota for around
2000 million years (Olson 2006, Knoll
2008). Cyanobacteria are believed to have
had a considerable effect on the formation
of the oxygen rich gas composition of
Earth’s atmosphere (Dismukes 2001, Paul
2008). This in turn had a profound effect
on the planet’s environmental conditions
such as the availability of nutrients and the
formation of the ozone layer. In addition,
they are also considered to be the origin of
eukaryotic chloroplasts (Raven and Allen
2003).
During their long evolutionary
history cyanobacteria have evolved a wide
spectrum of forms varying from unicellular
to filamentous morphology (Mur et al.
1999, Castenholtz 2001). Many of them
produce specialised cells called heterocytes
for the assimilation of molecular nitrogen
or resting cells called acinetes. In addition,
many cyanobacteria produce motile
filaments called hormogonia that function
as pioneers for starting new colonies away
from the main cyanobacterial biomass
(Castenholtz 2001).
Cyanobacteria are known to withstand
a wide range of environmental conditions
in terms of temperature, light conditions,
salinity and moisture. They can be found
in hot springs and in salt lakes, on desert
crusts or symbionts with lichens and are
an important part of both freshwater and
marine ecosystems (Whitton and Potts
2000, Oren 2000). We live beside the
cyanobacteria and exploit them for various
purposes ranging from oxygen sources
to rice field fertilizers (Oren 2000, van
Hove and Lejeune 2002). Unfortunately
this coexistence does not always occur
without problems, which has given the
cyanobacteria a somewhat infamous
reputation.
1.2 Cyanobacterial mass
occurrences
Perhaps the most familiar association that
people have with cyanobacteria is the mass
occurrences of planktonic cyanobacteria,
also known as the cyanobacterial water
blooms. During the mass occurrences
cyanobacterial cells can be spread out
into a large water mass and be quite
imperceptible (Mur et al. 1999, Oliver and
Ganf 2000, Huisman and Hulot 2005). In
calm weather the cells can then rapidly
concentrate onto the surface layer and
form a dense, floating cell matt. Mild wind
and currents can transport the water bloom
to shore. This increases the possibility of
humans and animals to come in contact
with the cyanobacterial mass. In addition,
cyanobacteria such as Planktothrix and
Cylindrospermopsis can form mass
occurrences several meters below the
water surface, which can hinder their
detection (Mur et al. 1999, Havens 2008,
Sivonen and Börner 2008).
1.2.1 Bloom forming cyanobacteria
The water blooms can be produced by
various cyanobacterial species within
1
Introduction
genera such as Anabaena, Aphanizomenon,
Cylindrospermopsis, Planktothrix,
Gloeotrichia, Microcystis and Nodularia
(Chorus et al. 2000, Oliver and Ganf
2000). In the tropics the blooms can prevail
all year round, whereas in the temperate
zones these water blooms usually occur
during late summer and early autumn,
subsiding during the cold and dark winter
period (Sivonen and Jones 1999, Oliver
and Ganf 2000). However, cyanobacterial
water blooms can persist through winter
also in temperate zones, as happened in
Finland during the early 1980’s on Lake
Vesijärvi and in the winter of 2008–2009
on Lake Saimaa (Keto and Tallberg 2000,
Kaakkois-Suomen ympäristökeskus
2009).
Cyanobacteria have various features
that help them to overcome other
phytoplankton species and form dense cell
masses in aquatic environments. Perhaps
the most important of these features
are the gas vesicles possessed by many
planktonic cyanobacterial species (Paerl
2000, Castenholtz 2001). These vesicles
can be used by cyanobacteria to regulate
their buoyancy to position themselves at
a depth favourable in terms of nutrients
and light. Another important feature in
aquatic environments where nitrogen
is often the growth limiting factor, is
the capability of many cyanobacteria
to fix atmospheric molecular nitrogen
(Castenholtz 2001). Other features thought
to enhance cyanobacterial survival and
competition status include features such
as siderochrome excretion for sequestering
iron, and the ability to utilize low photon
fluxes for photosynthesis.
1.3 Cyanobacterial toxins
Cyanobacteria produce a wide variety of
secondary metabolites, many of them with
so far unknown functions. The metabolites
2
are known to include several kinds of
compounds which are toxic to humans,
animals and plants. The toxin production
of aquatic, planktonic and benthic
cyanobacteria is the most studied and
the majority of the cyanobacterial water
blooms are shown to be toxic (Sivonen
and Börner 2008). However, terrestrial
cyanobacteria are also known to produce
toxins (Oksanen et al. 2004, Sivonen and
Börner 2008).
1.3.1 Hepatotoxins
The best characterised cyanobacterial
toxins are the hepatotoxic microcystins
and nodularins. They are the most
frequently found toxins in association
with cyanobacterial water blooms
(Sivonen and Börner 2008). Microcystins
are cyclic heptapeptides and nodularins
cyclic pentapeptides. Both microcystins
and nodularins contain an unusual Adda
amino acid ((2S,3S,8S,9S)-3-amino-9methoxy-2,6,8-trimethyl-10-phenyldeca4,6-dienoic acid), that has an important
role in their toxicity together with their
cyclic structure (Craig and Holmes 2000,
Sivonen and Börner 2008).
More than 80 microcystins variants
are known, whereas known variants of
nodularin are relatively rare (Sivonen
2009). Microcystins and nodularins are
produced nonribosomally by peptide
synthetase and polyketide synthase
complexes (Börner and Dittmann 2005,
Welker and von Döhren 2006). During
the active growth phase the toxins
are intracellular (Sivonen and Jones
1999). During cell death and lysis the
toxins are released into the surrounding
water. Microcystins are produced by
cyanobacteria which belong to the
genera such as Microcystis, Planktothrix/
Oscillatoria, Anabaena, Anabaenopsis,
Radiocystis, Synechococcus and Nostoc
Introduction
(Sivonen and Börner 2008). The
nodularins are produced by Nodularia.
The amounts and types of produced toxins
vary between different cyanobacterial
genera and species. In addition, the phase
of the cyanobacterial bloom and the size
of the cyanobacterial colonies may affect
toxin production (Kardinaal and Visser
2005).
Microcystins and nodularins
are inhibitors of eukaryotic protein
phosphatases 1 and 2A (Sivonen and
Börner 2008). They are water soluble
and are transported into cells mostly
by bile acid type transporters present in
hepatocytes and in the enterocytes of
the small intestine (Kuiper-Goodman et
al. 1999, Sivonen and Jones 1999). The
specific uptake system largely restricts
the effect of the toxins in mammals to the
liver, which gives them the classification
of hepatotoxins (Sivonen and Jones
1999). Cyanobacterial hepatotoxins cause
disruption of intracellular architecture
and a loss of cell-to-cell adhesion, which
leads to haemorrhage (Craig and Holmes
2000). Other acute effects attributed to the
cyanobacterial hepatotoxins may include
the generation of reactive oxygen species,
alteration of mitochondrial membrane
permeability and induction of cell
apoptosis (Humpage 2008).
The lethal dose of cyanobacterial
hepatotoxins varies depending on the route
of exposure as well as the hepatotoxin
variant and the exposed organism.
In mice the lethal dose, 50% (LD50)
of microcystin-LR by intraperitoneal
injection varies from around 25 to 150 μg
kg-1 body weight whereas the LD50 dose
by the oral route is 5000 μg kg-1 body
weight (Kuiper-Goodman et al. 1999, de
Figuiredo et al. 2004). In addition to acute
intoxication, cyanobacterial hepatotoxins
can cause adverse chronic health effects
such as the proposed tumour promotion,
by long term exposure to low toxin doses
(Kuiper-Goodman et al. 1999, Falconer
2008, Humpage 2008).
1.3.2 Neurotoxins
Both planktonic and benthic forms
of cyanobacteria produce several
kinds of neurotoxins. These toxins
include anatoxin-a and its homologue
homoanatoxin-a, anatoxin-a(S), saxitoxins
and β-N-methylamino-L-alanine (Sivonen
and Börner 2008). Anatoxin-a is a
secondary amine, which is produced by
species within the genera Anabaena,
Cylindrospermum, Aphanizomenon,
Planktothrix, Oscillatoria, Phormidium
and Arthrospira (Sivonen and Börner
2008). It is an alkaloid that binds to
neuronal nicotinic acetyl cholinesterase
receptors and causes nerve depolarisation
and muscle overstimulation (KuiperGoodman et al. 1999, Humpage 2008).
This can lead to health effects such as
staggering, gasping, convulsions and
respiratory failure (Humpage 2008).
Its homoanalogue homoanatoxin-a, is
produced by Oscillatoria/Planktothrix
and Raphidiopsis (Humpage 2008,
Sivonen and Börner 2008). Anatoxina(S) is an organophosphate produced by
Anabaena (Sivonen and Börner 2008), that
inhibits the enzyme acetyl cholinesterase
(Falconer 2008, Humpage 2008). The
resulting health effects are similar to those
caused by anatoxin-a, with the addition of
lacrimation, ataxia, diarrhoea, tremors and
hypersalivation, for which the (S) in the
toxin name stands for.
Saxitoxins are alkaloid neurotoxins
also known as paralytic shellfish poisons
(Sivonen and Börner 2008). They include
nonsulphated (saxitoxin and neosaxitoxin),
singly sulphated (gonyautoxins) and
doubly sulphated (C-toxins) compounds.
3
Introduction
Of these compounds, saxitoxin is the
most toxic. Saxitoxins were originally
identified in marine dinoflagellates. Of
the cyanobacteria they are produced
by Aphanizomenon, Anabaena,
Cylindrospermopsis, Lyngbya and
Planktothrix (Sivonen and Börner
2008). Saxitoxins affect the nerve axon
membranes by blocking sodium ion
channels, which causes changes in nerve
impulse generation (Kuiper-Goodman et
al. 1999). This can lead to symptoms such
as lip numbness, paralysis and respiratory
failure.
The acute LD50 dose of cyanobacterial
neurotoxins by intraperitoneal injection in
mice tests varies from about 10 μg kg-1
body weight for saxitoxin, to 200−250 μg
kg-1 body weight for anatoxin-a and for
homoanatoxin-a (Smith 2000). Saxitoxins
may accumulate in shellfish that form
an additional route of oral exposure
through ingestion and cases of acute
intoxication due saxitoxins in people
consuming shellfish have been described
(Kuiper-Goodman et al. 1999, Humpage
2008). However, the LD50 values by oral
exposure are several times higher that
of the intraperitoneal route. The chronic
effects of the cyanobacterial neurotoxins
are largely unknown (Kuiper-Goodman
et al. 1999, Falconer 2008, Humpage
2008). Some findings even indicate that
pre-exposure to saxitoxins may lower the
susceptibility to adverse health effects on
subsequent exposure to these toxins.
Quite recently, another neurotoxin,
the β-N-methylamino-L-alanine, originally
found in cycad seeds was shown to be
produced by various cyanobacterial
genera (Cox et al. 2005, Humpage 2008).
The β-N-methylamino-L-alanine has
been associated with neurodegenerative
disorders (Humpage 2008). It can be
bound by the proteins within the body,
4
which then may function as transporters
between organisms (Murch et al. 2004). In
addition, they can act as a reservoir of the
toxin, releasing it slowly during protein
metabolism.
1.3.3 Other toxins of cyanobacteria
Examples of other types of toxins or
toxic compounds of the cyanobacteria
are the cylindrospermopsin and
lipopolysaccharides (Sivonen and Jones
1999). Cytotoxic cylindrospermopsin
is a cyclic guanide alkaloid that is
produced by species within the genera
C y l i n d ro s p e r m o p s i s ,
Umezakia,
Aphanizomenon, Anabaena and
Raphidiopsis (Sivonen and Börner 2008).
It inhibits protein synthesis in general,
causing tissue injury in the liver, kidneys,
lungs, heart, adrenal glands, spleen and
thymus (Kuiper-Goodman et al. 1999,
Humpage 2008). Cylindrospermopsin
has also been shown to have genotoxic
effects, and in vivo studies indicate it to be
carcinogenic (Humpage 2008).
In contrast to the above
mentioned cyanobacterial toxins,
the lipopolysaccharides are cell wall
components of the cyanobacteria and
of Gram-negative bacteria in general.
The cyanobacterial lipopolysaccharides
seem have a lower toxicity than the
lipopolysaccharides of the Gram-negative
bacteria (Stewart et al. 2006a). Their
role as toxins is under study but they
are suspected to act as contact irritants
and a possible cause of gastro-enteritis
and adverse respiratory effects (KuiperGoodman et al. 1999, Anderson et al.
2002, Stewart et al. 2006a). The typical
exposure routes of humans to external
endotoxins include direct physical contact,
oral ingestion of endotoxin containing
matter, inhalation of endotoxic aerosols
and in some cases intravenous exposure.
Introduction
The most significant route of exposure in
association with cyanobacterial blooms
is probably by inhalation (Stewart et al.
2006a, Anderson et al. 2007).
1.3.4 Toxin analyses
The methods used to detect cyanobacterial
toxins vary depending on the toxin type
and whether the objective of the study
is in examining the toxin types, toxin
concentration or toxicity of the sample.
In the early studies, the mouse bioassay
was often used but its use has since
declined due to its non specificity, the
development of new detection methods
and ethical issues concerning animal
testing (Chu 2000, Sivonen 2000, Spoof
2005). Other bioassays, such as the desert
locust (Schistocerca gregaria) test for
saxitoxins and brine shrimp (Artemia
salina) test for neurotoxins, hepatotoxins
and cylindrospermopsin have also been
used (Chu 2000, Sivonen 2000, Metcalf et
al. 2002).
The bioassays have mostly been
replaced by a wide variety of chemical,
enzymatic and immunochemical assays
(Chu 2000, Sivonen 2000, Lawton
and Edwards 2008). The chemical
assays include methods such as highperformance liquid chromatography
(HPLC), mass spectrometry, thin-layer
chromatography and combinations
of these. The immunochemical
assays include methods such as
radioimmunoassay and modifications
of the enzyme-linked immunosorbent
assay (ELISA). The enzymatic assays
utilize protein phosphatase inhibition
assays for microcystins and nodularin
or acetylcholine esterase inhibition for
preliminary identification of anatoxina(S). The high-performance liquid
chromatography (HPLC) approach is often
used for the analyses of cyanobacterial
hepatotoxins, saxitoxins, anatoxin-a and
cylindrospermopsin (Spoof 2005, Metcalf
and Codd 2005a, b and c, Lawton and
Edwards 2008). The enzyme-linked
immunosorbent (ELISA) and protein
phosphatase inhibition assays are used
as relatively inexpensive and rapid
methods for analyses of cyanobacterial
hepatotoxins, whereas the acetylcholine
esterase inhibition assay is used for
screening of the putative presence of
anatoxin-a(S) (Ellman et al. 1961,
Chu 2000, Rapala et al. 2002a, Spoof
2005, Lawton and Edwards 2008).
Immunoassays, such as RIDASCREEN®
(R–Biopharm AG, Germany) and
Jellet Rapid Test (Jellett Rapid Testing
Ltd., Canada), are also available for
saxitoxins (Jellet et al. 2002, Lawton and
Edwards 2008). The lipopolysaccharide
concentrations can be measured using the
chromogenic Limulus amoebocyte lysate
assay (Metcalf and Codd 2005b).
The chemical, immunological and
enzymatic assays outcompete the bioassays
in accuracy, specificity and having a lesser
need for the test materials. However, they
have their own disadvantages such as a
high cost of the equipments, the need for
expertise, problems in sensitivity due to
structural variations within the studied
toxin types and the lack of available toxin
standards (Chu 2000, Sivonen 2000,
Lawton and Edwards 2008). Therefore,
bioassays such as the Artemia salina test
can still be useful as a relatively cheap
and easy option for cyanobacterial toxin
analyses, especially to screen samples for
the presence of different cyanobacterial
toxins types (Lahti et al. 1995, Sivonen
2000, McElhiney and Lawton 2005).
Bioassays are also needed in assessing the
acute and chronic toxicity of compounds
produced by cyanobacterial on organisms
such as mammals.
5
Introduction
1.3.5 Persistence of cyanobacterial
toxins
The persistence of the cyanobacterial
toxins varies depending on the toxin
type and whether the toxins are located
within or outwith the cyanobacterial
cells (Sivonen and Jones 1999, Chorus
et al. 2000). In addition, the chemical,
physiological and biological circumstances
affect the persistence of cyanobacterial
toxins. In general, the cyanobacterial
toxins are more stable in the dark than
in sunlight. The hydrolysing effect of
the sunlight can be potentiated by the
presence of cell pigments (Tsuji et al.
1994, Robertson et al. 1999). Other
chemical or physical factors that affect
the toxin breakdown include temperature,
pH, and the presence of oxidizing agents
such as ozone and chlorine (Sivonen and
Jones 1999, Chu 2000). However, the
most important method of removing the
cyanobacterial toxins in the environment
is probably biodegradation by microbes.
In general, the cyanobacterial
hepatotoxins microcystins and nodularins
are chemically more stable than the
neurotoxins or cylindrospermopsin. In the
dark they can remain toxic for months or
years and their toxicity is not destroyed
even by boiling (Sivonen and Jones 1999).
The persistence of cyanobacterial toxins
can be further assisted through protection
by the cyanobacterial cells. For example,
hepatotoxin containing cyanobacterial cell
scum found on shores can release toxins
into water (Jones et al. 1995). They can
also form another route of exposure to
the toxins through aerosols from toxic
cell matter. Therefore, the importance of
biodegradation of both cyanobacterial
cell mass and toxins is emphasized in the
removal of microcystins and nodularins
from the environment (Sivonen and Jones
1999). Biodegradation is also important
6
in the elimination of other cyanobacterial
toxin types, as could be seen in the
study of Rapala et al. (1994) in which
the anatoxin-a degradation depended on
biodegradation by microorganisms from
the sediments.
The majority of the biodegradation
studies on cyanobacterial toxins
have concentrated on hepatotoxins.
Biodegradation of the cyanobacterial
hepatotoxins have been detected in lake
and river water, lake sediments, sediments
of a water recharge facilities and in
sewage effluent waters (Sivonen and Jones
1999, Holst 2003). So far, the majority of
the isolated bacterial strains capable of
degrading cyanobacterial hepatotoxins
have belonged to the genus Sphingomonas
and to unidentified Betaproteobacteria
(Jones et al. 1994, Bourne et al. 1996 and
2001, Lahti et al. 1998, Park et al. 2001,
Saito et al. 2003, Saitou et al. 2003, Ishii
et al. 2004, Maryama et al. 2006, Ho et al.
2007).
The hepatotoxin degradation
mechanism of Sphingomonas has been
studied and it seems to include protein
products encoded by the genes mlrA,
mlrB, mlrC and mlrD (Bourne et al.
1996 and 2001, Saito et al. 2003, Ho
et al. 2007). Together they break down
the cyclic microcystin-LR structure into
a linear toxin by breaking the AddaArginine peptide bond and by breaking the
linear toxin into tetrapeptides and further
into smaller peptides and single amino
acids (Bourne et al. 1996 and 2001). The
hepatotoxin degradation mechanism in the
unidentified Betaproteobacteria strains
(Lahti et al. 1998) is unknown.
1.4 Aquatic cyanobacteria and
heterotrophic bacteria
Cyanobacteria and heterotrophic
bacteria are an important part of aquatic
Introduction
ecosystems. They have an invaluable
role in nutrient cycling in freshwater
and marine environments. At times
planktonic cyanobacteria can form the
majority of the phytoplankton biomass
and are responsible for most of the
plankton primary production (Paerl 2000,
Giovannoni and Stingl 2005, Eiler et al.
2006b). They are associated with various
kinds of heterotrophic bacteria (Eiler and
Bertilsson 2004, Kolmonen et al. 2004,
Eiler et al. 2006b, Tuomainen et al. 2006),
which use the carbon fixed by the primary
producers and form an important part of
the aquatic nutrient web.
1.4.1 Aquatic bacteria
Based on cultivation-independent studies,
the microbial plankton populations seem
to form habitat-specific clusters that
differ between marine and freshwater
environments (Hagström et al. 2002,
Zwart et al. 2002, Giovannoni and Stingl
2005). Cyanobacteria are detected from
freshwater and marine environments
in varying proportions. The major
bacterial divisions of freshwater
heterotrophic bacteria include Alphaand Betaproteobacteria, Actinobacteria,
Cytophaga-Flavobacterium-Bacteroidetes
and Verrucomicrobia (Glöckner et al.
2000, Zwart et al. 2002). In contrast, the
Betaproteobacteria form only a small
proportion of the marine heterotrophic
bacterial plankton, whereas the Gammaand Alphaproteobacteria form a majority
of the detected bacterial plankton of
the marine environments (Hagström et
al. 2002, Zwart et al. 2002, Rusch et al.
2007). Interfaces of these two aquatic
environment types such as estuaries and
coastal seas may contain bacterial groups
typical of both fresh and marine water
type. As a brackish water body the Baltic
Sea also largely falls into this intermediate
group of water bodies (Sivonen et al.
2007).
1.4.2 Interactions between
heterotrophic bacteria and
cyanobacteria
The abundance and taxonomic distribution
of the heterotrophic bacterioplankton
associated with cyanobacterial mass
occurrences can be affected by
several chemical, physiological and
biological factors such as nutrient,
light and temperature conditions, pH,
salinity and grazing. The presence and
metabolic activities of large amounts
of cyanobacterial cells can affect many
of these chemical, physiological and
biological factors (Paerl 2000, Wilson et
al. 2006, Havens 2008). The metabolic
activities such as photosynthesis
performed by cyanobacterial cells can
affect the oxygen and pH conditions of
the surrounding water and change the
amounts of available organic compounds
and nutrients as well as abundances
of antibacterial compounds (Paerl and
Pinckney 1996, Østensvik et al. 1998,
Casamatta and Wickstrom 2000, Oliver
and Ganf 2000, Kirkwood et al. 2006). For
example, many planktonic cyanobacterial
species can fix atmospheric nitrogen and
convert it into forms accessible to other
organisms (Oliver and Ganf 2000, Paerl
2000). The cyanobacterial cell mass also
offers additional particle surfaces to which
the heterotrophic bacteria can attach to
and provide protection by means of the
nutrient-rich polysaccharide sheaths of the
cyanobacteria (Paerl and Pinckney 1996,
Islam et al. 1999, Paerl 2000, Maryama et
al. 2003, Salomon et al. 2003, Eiler et al.
2006b).
In considering these factors, it is no
surprise that the cyanobacterial water
blooms seem to affect the amounts
7
Introduction
and composition of the planktonic
heterotrophic bacteria. The observed cell
concentrations of heterotrophic bacteria
can be substantially higher during and
immediately after cyanobacterial water
blooms than in their absence (Bouvy et al.
2001, Eiler and Bertilsson 2004 and 2007).
In the studies by Islam et al. (1999, 2002
and 2004) the presence of cyanobacteria
was found to prolong the cultivability of
Vibrio cholerae and the viable Vibrio cells
could be seen attached to cyanobacterial
cells long after they could no longer be
cultivated. In addition, in a study by Eiler
et al. (2007), dissolved organic matter
from the cyanobacterium Nodularia
spumigena increased the measured Vibrio
cholerae and Vibrio vulnificus abundances.
Specific attachment to the heterocytes,
cyanobacterial nitrogen fixing cells,
by heterotrophic bacteria has also been
detected, with indications that the attached
bacteria utilize amino acids produced by
the cyanobacteria (Paerl 1976, Paerl and
Kellar 1978). Furthermore, the study by
Kangatharalingam et al. (1991) showed
a positive relationship between the
chemotaxis of Aeromonas hydrophila
to cyanobacteria and the cyanobacterial
biomass. Positive chemotaxis to
cyanobacterial exudates has also been
seen in other bacteria co-occuring with
cyanobacteria (Casamatta and Wickstrom
2000). These studies indicate that
heterotrophic bacteria often benefit from
the presence of cyanobacteria. The fact
that many cyanobacterial bloom species
have not successfully been grown as
axenic cultures but seem to prefer the
presence of other bacteria indicates that
the benefit is in several cases mutual (Paerl
1996 and 2000).
The heterotrophic bacteria, in turn,
can inhibit or enhance the growth of
cyanobacteria and therefore affect the
8
formation and decline of cyanobacterial
mass occurrences. The known bacteria
that are able to affect the cyanobacterial
growth include species such as Cytophaga,
Shewanella, Streptomyces and Vibrio
strains (Yamamoto et al. 1998, Yoshikawa
et al. 2000, Rashidan and Bird 2001,
Manage et al. 2000, Salomon et al. 2003).
They can inhibit the cyanobacterial growth
by means such as competition for limiting
nutrients and by production of diffusible
lytic compounds (Yamamoto et al. 1998,
Rashidan and Bird 2001). The growth can
be enhanced by means such as production
of CO2, which cyanobacteria can use as
a carbon source or by the stimulation of
cyanobacterial photosynthetic and nitrogen
fixation activity through the reduction
of photosynthetic oxygen tension (Paerl
and Kellar 1978, Mouget et al. 1995,
Paerl and Pinckney 1996, Paerl 2000).
The cyanobacteria may also benefit from
the vitamins and remineralized nutrients
produced by the heterorophic bacteria
(Paerl and Pickney 1996, Simon et al.
2002) However, the exact mechanism
behind the cyanobacterial growth
inhibition or enhancement by heterotrophic
bacteria is often unknown.
1.5 Bacterial characterisation
Interpretation of the interactions between
bacteria and their role in the environment
requires knowledge on their characteristics
such as enzymatic and metabolic activities.
For studying the bacterial characteristics,
cultivable isolated bacterial strains
provide a valuable means compared with
the cultivation-independent methods
(Palleroni 1997, Zinder and Salyers 2001,
Roselló-Mora and Amann 2001). Thus,
the cultivation methods are an invaluable
asset of the microbiological and ecological
studies, even though they are laborious
and the detected bacteria are restricted to
Introduction
those supported by the chosen cultivation
conditions.
1.5.1 Isolation of bacteria
The isolated bacterial strains can vary
greatly depending on factors such as
the condition of the cells, incubation
temperature, incubation time, pH, the
surrounding oxygen levels and whether
a solid or liquid growth medium is used
(Zinder and Salyers 2001, Simu and
Hagström 2004, Cardenas and Tiedje
2008). In addition, the used growth media
may lack substances essential to the
desired bacteria or alternatively contain
them at toxic concentrations. For example,
rich growth media may contain nutrients
in concentrations toxic to aquatic bacteria
that are usually adapted to quite nutrient
poor conditions (Zinder and Salyers 2001).
For these bacteria a growth medium with
low nutrient content, such as sterilized
natural water or artificial medium such
as Reasoner’s 2A (R2A), would be more
suitable (Massa 1998, Zinder and Salyers
2001). However, nutrient rich growth
media may help to identify important
bacteria that might not be detected by
using only nutrient poor growth media.
Therefore, combinations of different
growth media and incubation conditions
should be used when endeavouring to
access as many of the cultivable bacteria
as possible.
1.5.2 Phenotypic analyses
Before the era of molecular methods,
bacterial classification was based on
required growth conditions, morphology
and physiological features of the bacteria
(Roselló-Mora and Amann 2001).
Analyses of these characteristics still
continue to play an important role in the
bacterial identification and information on
phenotypic characteristics is always needed
for describing a new taxon (Palleroni
1997, Roselló-Mora and Amann 2001). In
addition, informativity of the taxonomic
assignment of new bacterial isolates or
16S rRNA gene clones is largely based
on the known characteristics of the close
relatives of the studied bacteria (Palleroni
1997).
The analyses of cell morphology
include microscopy for studying cell
features such as size and shape, Gram
staining and presence of flagellas
(Roselló-Mora and Amann 2001). In
addition, the morphology includes the
characters such as colour and size of the
bacterial colonies. Of the biochemical
components, characteristics such as fatty
acid composition and protein profile
of the cells and cell wall composition
of the Gram-positive bacteria are often
investigated.
Analyses for enzymatic activities and
growth requirements are usually performed
using commercial test kits, that contain
several tests in miniature form. These
include kits such as the widely used API
tests (bioMèrieux, Marcy-l’Etoile, France)
and the Biolog (Biolog, Inc., Hayward,
CA, USA) for identification of bacteria of
specific metabolic types. For example, the
API 20 NE test kit for the identification
of non-fastidious, non-enteric, Gramnegative rods, such as Pseudomonas,
Flavobacterium and Vibrio, and Biolog
tests versions for both Gram-negative
and Gram-positive bacteria are available
(Biolog 2001a and b, bioMèrieux 2009,
Stager and Davis 1992, Truu et al. 1999,
Awong-Talylor et al. 2008). The API ZYM
test kit and the ZymProfiler® fluorogenic
analysis designed by Vepsäläinen et al.
(2001) can be used for the screening of
enzymatic activities of bacteria from
purified or complex samples (bioMèrieux
2009, Tharagonnet et al. 1977, Allen et
9
Introduction
al. 2002, Niemi et al. 2004). In addition,
noncommercial methods such as the
Aeromonas hydrophila (AH)-medium tube
test by Kaper et al. (1979) can be used for
examining several physiological features
simultaneously. The information obtained
from cultivated, pure bacterial strains offer
new reference points for the large scale
studies, which relay on the data obtained
by molecular methods, and help to clarify
the roles of different bacterial groups in
the environment.
1.5.3 Molecular analyses in taxonomic
identification of bacteria
The use of DNA based methods in
taxonomic identification of bacteria
began with the analyses of genomic
guanine-cytosine content and DNA-DNA
hybridization of the whole genomic DNA
(Roselló-Mora and Amann 2001). These
methods offer a valuable asset in closer
taxonomic assessment of bacteria and are
still required for describing a new taxon.
However, the routine use of polymerase
chain reaction (PCR) and DNA sequencing
have made the analyses of the rRNA gene
sequences an inseparable part of bacterial
taxonomic analyses. Sequence analyses
of the universal and conserved 16S rRNA
gene are especially used as relatively
cheap and quick methods of taxonomic
identification of bacteria (Roselló-Mora
and Amann 2001, Clarridge 2004). The
popularity of the16S rRNA analyses is
based partly on the rapidly growing 16S
rRNA sequence databases and a wide
variety of quick search tools provided
freely on the internet. These include tools
such as the basic local alignment search
tool (BLAST) provided by the National
Center for Biotechnology Information
(NCBI) and the taxonomic classifier and
sequence match tools provided by the
Ribosomal Database Project II.
10
A wide variety of methods included
in both free and commercial software
packages, such as the commonly used
phylogenetic analyses using parsimony
(PAUP, D. L. Swofford, Sinauer
Associates, Sunderland, MA, USA) and
phylogeny inference package (PHYLIP,
Felsenstein 2005), are also available for
analyses of the phylogenetic relationships.
The phylogenetic relationships between
bacteria are often visualized by using
phylogenetic trees generated by methods
such as neighbour-joining, unweighted
pair group method with arithmetic
averages (UPGMA), maximum parsimony
or maximum likelihood (Nei and Kumar
2000b, c and d, Felsenstein 2004b).
The neighbour-joining method and
unweighted pair group method with
arithmetic averages (UPGMA) are based
on distance matrixes calculated from
sequence distances using models such as
the Kimura or the F84 model (Nei and
Kumar 2000b, Felsenstein 2004b). In
contrast, the maximum parsimony and
maximum likelihood methods are based on
the actual sequence data (Nei and Kumar
2000c and d). In addition to the tree
construction strategies, the methods differ
in the required computer time. Therefore,
the choice of method has often been the
computationally efficient neighbourjoining, especially when analysing large
datasets. In the maximum parsimony
method, the needed computer time can be
limited by choosing the heuristic search
option instead of exhaustive search (Nei
and Kumar 2000d). The reliability of
the obtained trees can be assessed by a
confidence test such as bootstrapping, and
by comparing the trees obtained by using
different tree building methods (Nei and
Kumar 2000a, Felsenstein 2004a).
Introduction
1.5.4 Genetic analyses in assessment of
physiological features
Since the physiological features of the
bacteria are ultimately based on their gene
content, molecular methods such as PCR
with gene specific primers can also be used
to assess putative physiological features
of the bacteria. For example, the virulence
potential of Aeromonas strains has been
assessed by screening for virulence genes
(e.g. Kingombe et al. 1999, Albert et al.
2000, Chacón et al. 2003, Sen and Rodgers
2004). The virulence of the Aeromonas
has been suggested to be multifactorial
and to depend on several different factors
such as the production of cytotoxic and
cytotonic enterotoxins, haemolysin, lipase
and elastase and the ability to move by
flagella (Janda 1991, Sen and Rodgers
2004, Galindo et al. 2006). PCR primers
have been designed for many of the genes
that encode the virulence features and the
presence of such genes has been screened
for in both clinical and environmental
Aeromonas strains (e.g. Chacón et al.
2003, Sen and Rodgers 2004). However,
to writer’s knowledge, studies about
Aeromonas strains associated with
cyanobacterial water blooms have not been
performed and the virulence potential of
such Aeromonas in general is still largely
unknown.
1.5.5 Statistical analyses of multivariate
data
Possible patterns and associations of the
studied variables, such as physiological
characteristics, genetic composition or
the isolation source of the bacteria, can be
examined and visualized using numerical
or statistical analyses. These include
methods such as the principal coordinate
analysis, discriminant, regression or
correlation analyses (Legendre and
Legendre 1998a and b, Zuur et al. 2007).
The suitability of the statistical methods
for analysing the measured variables
differs according to several factors such
as whether or not a quantitative form and
normal distribution of data are required.
Therefore, for example the chosen distance
or dissimilatory measure can considerably
affect the results of the analysis.
Many of the available methods are
based on a specific distance measure,
which can limit their usefulness for
analysing the studied data (Anderson and
Willis 2003). In contrast, the programs
for the principal coordinate analysis (also
called metric multidimensional scaling),
canonical discriminant and correlation
analyses or the multiple regression with
forward selection analysis by Anderson
(2003a and b, 2004) allow the distance
or dissimilarity measure of the analysis
to be chosen by the user. This enables the
analyses included in the programs to be
adjusted for a wide range of variable sets
ranging from DNA sequence data to often
zero inflated species abundance data. In
addition, the methods are suitable for data
in which the number of observations far
exceeds the number of variables.
The analyses included in the principal
coordinate analysis program (PCO)
can be used to visualize the ordination
of data on the basis of their calculated
distances such as grouping of bacterial
strains according to their 16S rRNA
sequence distances (Anderson 2003b).
The canonical discriminant and correlation
analyses included in the canonical analysis
of principal coordinates program (CAP),
can be used to uncover patterns in the
multivariate data and to test a priori
hypotheses of differences among groups
(Anderson and Robinson 2003, Anderson
and Willis 2003). The regression analyses
included in the distance-based multivariate
analysis for a linear model using forward
11
Introduction
selection program (DISTLMf) can be used
to analyse hypotheses on the relationship
between chosen response variables and
explanatory variables (McArdle and
Anderson 2001, Anderson 2003a). The
methods can also be combined, in order
to select the most notable variables for the
canonical discriminant analysis.
1.6 Problems associated with
cyanobacterial water blooms
Eutrofication of aquatic systems has
lead to the worldwide enforcement of
cyanobacterial mass occurrences and
the increasing problems caused by
them (Bartram et al. 1999, Paerl 2008).
Cyanobacterial water blooms can cause
various kinds of nuisances. As a relatively
mild consequence the cyanobacterial cell
mass can produce unpleasant odours and
taste to water but their effects can be much
more profound and damaging (Falconer
et al. 1999). Large cyanobacterial masses
can change the physical and chemical
features of a water body, such as lighting
and the availability of growth limiting
nutrients, thus causing changes to the
aquatic species composition and nutrient
web (Sivonen 2000, Fournie et al. 2008).
Adverse health effects in humans and
poisonings of cattle and wildlife such as
fish, birds and muskrats associated with
toxic cyanobacterial blooms in natural
water bodies are also often reported
(Kuiper-Goodman et al. 1999, Stewart
2006b, Stewart et al. 2008, Sivonen 2009).
In addition, insufficient drinking water
treatment of source water contaminated
by cyanobacterial mass occurrence has
caused adverse human health effects
(Kuiper-Goodman et al. 1999, Falconer
and Humpage 2005).
12
1.6.1 Adverse human health effects
associated with cyanobacterial water
blooms
One of the problems associated with
the cyanobacterial water blooms is the
adverse human health effects they can
cause. The health effects associated with
cyanobacterial mass occurrences vary from
mild to fatal and from chronic to acute
(Kuiper-Goodman et al. 1999, Stewart
2006a and b). Typical routes of exposure
are direct physical contact with blooms,
ingestion of water containing bloom matter
or by exposure to the aerosols emanating
from the bloom. Possible consequences of
exposure to a cyanobacterial water bloom
can be quite varied and include effects such
as skin irritation, fever, headache, nausea,
vomiting, muscular pains, gastrointestinal,
neurological and liver symptoms, and
symptoms of eyes, nose, ears and throat.
The various kinds of toxins
cyanobacteria produce as secondary
metabolites are often suspected, and in
some cases proven to be the source of
the adverse health effects. The severity
of the effects depends on many factors
such as the magnitude and length of the
exposure, the type and concentration of
the cyanobacteria and their toxins, the type
of contact, the age and the health status of
the patient (Kuiper-Goodman et al. 1999,
Salmela et al. 2001, Hoppu et al. 2002,
Stewart 2006b). However, the specific
cause of the health effects often remains
unclear, due to the lack of sufficient
sample data and epidemiological studies
on the reported cases (Bartram et al. 1999,
Stewart 2006b, Pilotto 2008).
Despite evidence of the close
interactions between cyanobacteria
and heterotrophic bacteria, the possible
influence of these associations have
rarely been studied when assessing the
health risks posed by the blooms (Chorus
Introduction
et al. 2000, Stewart et al. 2006b). For
example, the assessment of human health
risks associated with cyanobacterial
water blooms has been strongly focused
on cyanobacteria and their toxins, even
though many of the detected human health
effects, such as skin- and gastrointestinal
problems, could also have been caused by
heterotrophic bacteria.
Members of the genera such as
Aeromonas and Vibrio have been
associated with adverse health effects and
are indigenous to aquatic environments
worldwide (Thompson et al. 2004, MartinCarnahan and Joseph 2005). For example,
A. caviae, A. eucrenophila, A. hydrophila,
A. jandaei, A. media, A. popoffii, A.
sobria and A. veronii are found in aquatic
environments (Hänninen and Siitonen
1995, Borrell et al. 1998, Martin-Carnahan
and Joseph 2005). The highest Aeromonas
concentration in water environments are
usually reached during warm seasons
when the temperatures are also optimal
for growth of most cyanobacteria, and
recreational use of water bodies is most
common (Mur et al. 1999, Castenholz
2001, Farmer et al. 2006, Martin-Carnahan
and Joseph 2005).
In clinical samples the A. caviae,
A. veronii and A. hydrophila species are
the most prevalent Aeromonas detected
(Janda and Abbot 1998, Figueras 2005,
Lamy et al. 2009). Other Aeromonas
found in human clinical samples include
species such as A. media, A. jandaei,
mesophilic A. salmonicida, A. sobria,
A. allosaccharophila, A. popoffii and A.
eucrenophila. The adverse human health
effects associated with Aeromonas include
conditions such as diarrhoea, wound
infection, bacteraemia, urinary tract
infection, pneumonia and fever (Janda
1991, Janda and Abbot 1998, Figueras
2005). The mortality to Aeromonas
infections in risk groups in humans such
as immunocompromized persons, children
and the elderly can be high, 25−75 %
(Janda and Abbott 1998). Underlying
diseases of malignancy, hepatobiliary
disease and diabetes can also increase
the susceptibility to Aeromonas infection
and severity of the obtained illness
(Figueras 2005). Many of the Aeromonas
species are resistant to several antibiotics
and infections caused by them can
progress quite rapidly and with serious
consequences, even among persons with
no underlying diseases (Figueras 2005,
Martin-Carnahan and Joseph 2005).
Vibrio cholerae is best known for the
cholera epidemics and pandemics that the
strains of the serogroups O1 and O139
can cause (Faruque et al.1998, Leclerc
et al. 2002). However, other V. cholerae
strains and strains of V. parahaemolyticus
and V. vulnificus are also associated with
human infections such as gastroenteritis,
skin infections, wound infections and
septicaemia (Thompson et al. 2004,
Lukinmaa et al. 2006).
In addition, cyanobacteria together
with heterotrophic bacteria may represent
an increased health risk. The infection
susceptibility of persons exposed to
cyanobacterial water blooms could
be lowered by the adverse effects
of the cyanobacterial toxins. The
lipopolysaccharides of the Gram-negative
bacteria, in turn, inhibit glutathione
S-transferase activity, which is important
in detoxifying of microcystins, and
may increase the risk posed by the
cyanobacterial hepatotoxins (Best et al.
2002, Rapala et al. 2002b). Therefore,
studies on the heterotrophic bacteria
associated with planktonic cyanobacteria
could provide valuable information for the
risk assessment of these blooms.
13
Introduction
1.6.2 Cyanobacterial water blooms and
drinking water
The key to microbiologically, chemically
and radiologically safe drinking water
lies in the use of the combination of
different treatment and monitoring steps,
to ensure sufficient water quality despite
the normal performance fluctuations of
individual stages (LeChevallier and Au
2004b, WHO 2008c and f). This multiple
barrier approach begins from source water
protection and ends at the protection of the
distribution system (LeChevallier and Au
2004b).
Cyanobacterial blooms can cause
problems for drinking water treatment
all the way from the water source to the
drinking water distribution (Hrudey
et al. 1999, Svrcek and Smith 2004).
Toxic cyanobacteria are listed among
emerging pathogens that may contribute to
waterborne diseases and the World Health
Organization (WHO) has proposed a
lifetime safe consumption level of 1 μg l-1
for the microcystin-LR in drinking water
(Falconer et al. 1999, Hunter et al. 2003).
Studies on the effects of cyanobacteria
on the drinking water quality have
concentrated mainly on the removal and
inactivation of cyanobacterial toxins,
in most cases microcystins, by specific
treatment procedures (Kuiper-Goodman
et al. 1999, Lawton and Robertson 1999,
Hitzfeld et al. 2000, Svrcek and Smith
2004). Studies on operational water
treatment plants are also largely centred
on microcystins, and investigations on
other factors such as the removal of
cyanobacterial neurotoxins, phytoplankton
cells and endotoxins are few (Lepistö et
al. 1994, Lambert et al. 1996, Lahti et
al. 2001, Karner et al. 2001, Rapala et al.
2002b, Hoeger et al. 2005). Therefore, the
overall water purification efficiency of
different treatment combinations in a real
14
life situation during a cyanobacterial water
bloom in the source water can be hard to
assess.
1.6.3 Cyanobacterial toxins and the
drinking water treatment
If surface water is used, the water
treatment steps usually include
coagulation, flocculation and clarification
(by sedimentation or flotation) followed
by filtration and disinfection (LeChevallier
and Au 2004d). The effectiveness of
different treatment procedures on the
removal of cyanobacterial toxins has
been assessed but the available data are
still quite limited (Lawton and Robertson
1999, Hitzfeld et al. 2000, Svrcek and
Smith 2004). Most of the studies have
concentrated on the removal of the
chemically stable hepatotoxins. However,
different cyanobacterial toxins are
removed with varying degree of efficiency
and the efficient removal of hepatotoxins
does not guarantee removal of other
types of cyanobacterial toxins (Hitzfeld
et al. 2000, Svrcek and Smith 2004). For
example, ozonation of water can efficiently
remove cyanobacterial hepatotoxins and
anatoxin-a but is quite ineffective against
saxitoxins (Lawton and Robertson 1999,
Hitzfeld et al. 2000, Rositano et al. 2001,
Svrcek and Smith 2004).
The common factor affecting the
efficiency of removal of the cyanobacterial
toxins in many of the treatment steps is
whether the toxins remain inside or are
released outside the cyanobacterial cells
(Hitzfeld et al. 2000, Svrcek and Smith
2004). The coagulation, clarification and
filtration steps are used mainly to remove
particulate matter such as microbial cells
(LeChevallier and Au 2004d, WHO
2008b). By detaining the cyanobacterial
cells, these water treatment steps also
efficiently remove cyanobacterial toxins
Introduction
within those cells (Hitzfeld et al. 2000,
Drikas et al. 2001, Svrcek and Smith
2004). In contrast, coagulation and
clarification are quite inefficient against
toxins already released from the cells into
water. Filtration, especially with slow sand
filters that contain a biological layer, may
also help to remove the dissolved toxins
by biological degradation (Svrcek and
Smith 2004, LeChevallier and Au 2004d,
Ho et al. 2006, WHO 2008b).
Disinfection procedures such as
chlorination and ozonation destroy
cyanobacterial toxins outside the cells
more efficiently than when the toxins are
protected by intact cell structures (Hitzfeld
et al. 2000, Svrcek and Smith 2004). The
primary aim of disinfection is to inactivate
viable microbes and the concentrations
and contact times used in disinfection are
optimized for this purpose (LeChevallier
and Au 2004a, WHO 2008b). In addition,
compounds such as ozone can be used
as preoxidants to aid the coagulation and
filtration processes (Widrig et al. 1996,
Yukselen et al. 2006, Chen et al. 2009, Li
et al 2009). Ozonation within the range
of normal disinfectant concentrations and
contact times used in water treatment is
quite efficient against most toxin types
(Hrudey et al. 1999, Lawton and Robertson
1999, Hitzfeld et al. 2000, Rositano et al.
2001, Svrcek and Smith 2004, Westrick
2008). The efficiency of chlorination
depends on the chlorine compound used
and higher concentrations and longer
contact times than normally used are often
required for effective toxin inactivation.
Since the source water can contain
both cell retained toxins and extracellular
toxins, the water treatment process
should be able to cope with both of them.
In addition, when designing the water
treatment train, it should be considered
whether the cell retained or extracellular
toxins are the main target of the toxin
removal, so that the chosen treatment steps
will not counteract with each other (Lawton
and Robertson 1999, Svrcek and Smith
2004). Various methods and application are
available for each of the usual purification
steps. For example, in the filtration step
rapid gravity and pressure filters or slow
sand filters can be used (Svrcek and Smith
2004, LeChevallier and Au 2004d, WHO
2008b). Additional steps such as pretreatment of the water with bank filtration
or precipitation of calcium and magnesium
by adjusting the pH of water with lime,
can also be used as a part of the treatment
process (LeChevallier and Au 2004d,
WHO 2008b). The removal of organic
compounds, such as the cyanobacterial
toxins, can be further improved by using
granular-activated carbon or powdered
activated carbon to adsorb them (Cook
and Newcombe 2002, Logsdon et al.
2002, Svrcek and Smith 2004). Efficient
biodegradation of microcystins has also
been detected during granular activated
carbon filtration, indicating that both
adsorption and biodegradation contribute
to the removal and inactivation of the
toxins (Wang et al. 2007).
1.6.4 Combined effects of
cyanobacterial water blooms and
heterotrophic bacteria on the drinking
water quality
In addition to the produced toxins,
cyanobacterial water blooms in general
can cause problems for the water treatment
and water quality by the changing physical
and chemical conditions that affect the
overall functioning of the water treatment
processes. They also produce compounds,
such as geosmin, that can cause unpleasant
taste and odour to the water (Peterson
et al. 1995, Chow et al. 1998, Falconer
et al. 1999). Cyanobacterial water
15
Introduction
blooms in the source water can cause
substantial increases in the amounts of
organic particles and overall amounts of
organic compounds that enter the water
treatment system (Hrudey et al. 1999).
This additional organic matter can cause
problems for the water quality, if the
treatment process is not sufficiently quickly
adjusted to match the new situation. The
adjustment can be made, for example, by
changing the source water intake depth, by
optimizing the coagulation process, or by
utilizing activated carbon for the removal
of organic substances or by increasing
the amounts of disinfectant used (Bursill
2001, LeChevallier and Au 2004a and d,
Svrcek and smith 2004, Westric 2008).
In addition, the CO 2 consumption by
the photosynthetic reactions within the
cyanobacterial bloom can elevate the raw
water pH, which in turn can affect several
water treatment steps, especially those
of coagulation and ozonation (Chow et
al. 1998, Hitzfeld et al. 2000, Briley and
Knappe 2002, WHO 2008b, Havens 2008,
Westrick 2008).
During cyanobacterial mass
occurrences, when a treatment system is
struggling at its uppermost limit, higher
than normal amounts of heterotrophic
bacterial and phytoplankton cells may pass
through the treatment system (Lepistö et
al. 1994). This may increase direct health
risks to the water consumers (WHO
2008d). The viable bacteria and organic
matter passing through the water treatment
system may also cause problems to the
water quality by biofilm formation in the
distribution system, which can affect the
water quality both directly and indirectly
(Rusin 1997, Percival and Walker 1999,
WHO 2008a, c and e). Biofilms can affect
the taste and odour as well the colour of
the water and in some occasions function
as a shelter for putative pathogens, such as
16
Legionella, Mycobacterium, Aeromonas
or Pseudomonas (Percival and Walker
1999, Theron and Cloete 2002, Berry et
al. 2006, WHO 2008c and e). The biofilms
offer protection from the effects of the
disinfectants to the organisms within them
(Percival and Walker 1999, Theron and
Cloete 2002, Berry et al. 2006,WHO 2008a
and e). In addition, the interaction between
biofilms and disinfectants can lead to the
formation of harmful compounds such
as nitrite. The interactions between the
materials of the distribution system and the
bacterial cells as well as the compounds,
such as acids, produced by the microbial
metabolism can lead to substantial
corrosion of the distribution system
(Percival and Walker 1999). This causes
problems to both the water quality and
the condition of the distribution system,
which may have significant economic
consequences. The lipopolysaccharides of
the Gram-negative heterotrophic bacteria
may also increase the risk posed by the
cyanobacterial hepatotoxins that may pass
through the water treatment process (Best
et al. 2002, Rapala et al. 2002b). Therefore
the assessment of risks posed by the
cyanobacterial water blooms to drinking
water safety should cover the possible
combined effects of cyanobacteria and
heterotrophic bacteria.
As a counterbalance to their negative
effects, the heterotrophic bacteria can
also provide a means to control the
risks posed by cyanobacterial water
blooms to the drinking water safety and
quality. In a study by Ho et al. (2006)
microcystins were shown to be removed
from water through a process of biological
degradation in sand filters. Furthermore,
a Sphingopyxis strain that degraded
microcystins was isolated from such a
filter (Ho et al. 2007). The studies showed
that the heterotrophic bacteria adapted to
Introduction
metabolize the cyanobacterial hepatotoxins
can also contribute to the removal of
the toxins from water during the water
treatment process. Such bacteria might be
utilized in the removal of the toxins in the
drinking water treatment, for example as
a part of the biological layer of the slow
sand filters. In addition, cyanobacterial
growth inhibiting bacteria have been
proposed to be used as biological control
agents of cyanobacterial blooms (Rashidan
and Bird 2000). Therefore, the overall
risks of cyanobacterial water blooms to
the drinking water quality could perhaps
be reduced by utilizing such bacteria in
controlling cyanobacterial blooms in
drinking water reservoirs. However, more
information on the characteristics of such
bacteria and on their interactions with
cyanobacteria are needed for safe and
efficient use of such applications.
17
Aims of the Study
2. AIMS OF THE STUDY
Cyanobacterial mass occurrences, the water blooms, frequently cause problems for
recreational and potable use of water. The factors that affect the health risks associated
with cyanobacterial water blooms are in many respects still unclear. In addition, the
studies that assess the overall water treatment efficiency of operating treatment plants
encumbered with cyanobacterial mass occurrences are rare. The risk assessment
concerning the cyanobacterial blooms has largely been focused on the cyanobacteria
and their toxins. However, the water blooms are associated with diverse communities
of heterotrophic bacteria. These bacteria may, for their part, affect the development
and decline of the water blooms and also contribute to the health problems caused
by the blooms, but little is known about them. The studies concerning these bacteria
have mainly been based on cultivation-independent molecular methods that provide no
bacterial isolates for closer study.
The aims of this study were:
- to examine the community of cultivable heterotrophic bacteria associated with the
cyanobacterial mass occurrences using taxonomic and phenotypic characterisation of
bacterial isolates (I).
- to assess the virulence potential of Aeromonas spp. strains isolated from cyanobacterial
water blooms, by studying the presence of six virulence gene types specific to
Aeromonas (II).
- to study the genetic and phenotypic characteristics of previously isolated bacterial
strains shown to be able to degrade cyanobacterial hepatotoxins (III).
- to assess the water purification efficiency of a modern drinking water treatment process
at an operating water treatment plant, during mass occurrence of cyanobacteria in the
source water (IV).
18
Materials and Methods
3. MATERIALS AND METHODS
3.1 Water samples and isolated bacterial strains
Water samples were collected from Finnish lakes, rivers or ponds and from the Baltic Sea
(I, II, IV) and from treated drinking water of water treatment plants (I, IV). The sample
sources were selected based on increased cyanobacterial occurrence in the water bodies
or in the water source utilized for producing the drinking water. Samples were also
collected from sites where a cyanobacterial water bloom was suspected to have caused
human health symptoms (II). The water samples and bacterial strains isolated from
the samples are identified in the original articles (I, II, IV). In addition, 13 previously
isolated hepatotoxin degrading Betaproteobacteria strains were studied (III).
3.2 Methods
The methods used in this study are listed in table 1. A more detailed description of the
methods are given in the original papers (I−IV)
Table 1: Methods used in the study
Method
Sampling and strain isolation
Telephone interviews for collecting data on adverse human health effects
Phytoplankton identification and biomass analysis
Gram staining
Flagella staining
API NE and API ZYM tests
ZymProfiler® test
GN MicroPlate test
Fatty acid analysis by gas-liquid chromatography (GLC)
Bacterioclorophyll a analysis by spectrophotometry
Oxidase and catalase tests
Aeromonas hydrophila (AH) medium tests
Motility on casitone-yeast extract agar
Haemolytic activity on blood agar
Testing of the effects of the isolates on cyanobacterial growth
Hepatotoxins by enzyme-linked immunosorbent assay (ELISA)
Hepatotoxins by protein phosphatase inhibition assay
Endotoxins by kinetic chromogenic Limulus amoebocyte lysate assay
Overall toxicity by Artemia salina bioassay
Hepatotoxin degradation test
Anatoxin-a by high-performance liquid chromatography (HPLC)
Saxitoxins by Jellett Rapid paralytic shellfish poisons test strips
Putative presence of anatoxin-a(S) by acetylcholine esterase inhibition test
DNA extraction from bacterial strains
16S rRNA gene amplification and sequencing
Genomic guanine-cytosine content by high-performance liquid chromatography
(HPLC)
Taxonomic classification by basic local alignment search tool (BLAST)
Paper
I, II, IVa
II
IV
I, II, III, IV
III
III
III
III
III
III
II, III
II
III
I
I
II, IV
III, IV
II, IV
IV
III
II, IVb
II, IVc
II, IVd
I, II, III, IVe
I, II, III, IVf
III
I, III, IV
19
Materials and Methods
Table 1 cont.
Method
Paper
Taxonomic classification by taxonomic classifier and sequence match tools
I, II
16S rRNA gene sequence similarity analysis by BioEdit
II, III
Phylogenetic analysis with neighbour-joining and maximum parsimony criteria III
II
Visualization of a priori grouping by principal coordinate analysis
II
Screening of Aeromonas virulence genes by PCR
Multivariate multiple regression analysis
II
Canonical discriminant analysis
II
a
Strain isolation was done as described in I.
b
The presence of anatoxin-a, homoanatoxin-a, and their epoxy and dihydro degradation products
were analysed as described by James et al. (1997).
c
The presence of saxitoxins was analysed using the commercial Jellett Rapid paralytic shellfish
poisons test strips (Jellett et al. 2002).
d
The putative presence of anatoxin-a(S) was analysed as described by Ellman et al. (1961).
e
The DNA extraction was executed as described in III.
f
Sequencing was carried out at the Institute of Biotechnology of the University of Helsinki.
20
Results
4. RESULTS
4.1 Heterotrophic bacteria
associated with cyanobacteria
In order to study the heterotrophic bacteria
in association with cyanobacteria, over
600 bacterial strains were isolated.
These strains originated from freshwater,
brackish water or treated drinking water
samples, which were obtained from sites
with increased cyanobacterial occurrence
in the water bodies or in the water source
of the drinking water. Chosen subsets of
the strains were studied for characteristics
such as a set of virulence genes in 176
Aeromonas spp. strains, or for their effect
on cyanobacterial growth in 183 strains
of various bacterial genera. In addition,
13 cyanobacterial hepatotoxin degrading
bacteria strains previously isolated from
Finnish lakes with frequent cyanobacterial
occurrence were characterised.
4.1.1 Taxonomic assignment of the
isolated bacterial strains
The isolated strains represented bacteria
from five different phyla, eight different
classes, and 57 different genera based
on analyses of their partial (397–905
bp) 16S rRNA gene sequences (Fig. 1,
Appendix 1; I; II; IV). The majority (82%)
of the strains were assigned to Gamma-,
Alpha- and Betaproteobacteria classes,
whereas Actinobacteria, Flavobacteria,
Sphingobacteria and Bacilli formed 18%
of the isolated strains. The Alpha- and
Betaproteobacteria were both represented
by 13 different genera, and Actinobacteria,
Flavobacteria, Sphingobacteria, Bacilli
and Gammaproteobacteria by 3–9 genera.
The one strain representing the class
Deinococci belonged to Deinococcus.
In addition, 21 of the isolated strains
could be assigned only at family level
and 3 strains only to class level above
the confidence threshold (80%) that
was set as the limit for the taxonomic
assignment by the Ribosomal Database
Project II classifier (Fig 1, Appendix 1; I:
Table 1, supplementary data; II: Table 3,
supplementary data). The isolated strains
also included several of those that had
no close relatives (maximum identity
values <98.7% by basic local alignment
search tool (BLAST) analysis) among
data base sequences of either the 16S
rRNA genes of cultivated bacterial strains
or of uncultivated bacteria (I: Table 2).
Therefore, the strains represented a diverse
group of heterotrophic bacteria, including
previously undescribed bacterial species.
4.1.2 Bacteria degrading cyanobacterial hepatotoxins: Paucibacter
toxinivorans gen. nov. sp. nov.
The previously isolated 13 Betaproteobacteria strains were chosen for
characterization due to shown ability
to degrade cyanobacterial hepatotoxins
microcystins and nodularin, which had
been tested mainly using one microcystin
variant and with two of the strains also
using nodularin (III). Five of the strains
were tested for the ability to degrade of
nodularin of the Nodularia 790 strain and
microcystins LR and YR (leucine and
arginine (LR) or tyrosine and arginine
(YR) as the aminoacids 2 and 4) of
the Microcystis 764 strain (Appendix
1; III). All five strains degraded both
hepatotoxin types, as measured by the
protein phosphatase inhibition analysis.
Nodularin was degraded at the range of
maximum rates from 3 to 560 fg cell-1 h-1
and the microcystins LR and YR at the
maximum rates from 2 to 30 fg cell-1 h-1.
The results implied that the strains have
a stronger ability to metabolize nodularin
than microcystins.
21
Results
The taxonomy and physiological
characteristics of the 13 strains were
studied using molecular and biochemical
analyses. The closest relatives of the
strains, based on nearly complete (1384–
1444 bp) 16S rRNA gene sequences, were
the Betaproteobacteria strains R-8875
(AJ440994, 97.0–97.1% similarity) and
CD12 (AF479324, 96.5–96.8%), Mitsuaria
chitosanitabida (AB006851, 96.1–96.8%,
formerly Matsuebacter chitosanotabidus)
and Roseateles depolymerans (AB003623
and AB003625, 95.7–96.3%; III: Fig. 1).
Based on the 16S rRNA gene sequence
similarities, the 13 strains could be
assigned only at the family Incertae cedis
5 of the order Burkholderiales, implying
that they represented a new bacterial
genus.
The strains were oxidase positive
and weakly catalase positive. All strains
were strongly or clearly positive for
alkaline phosphatase, chymotrypsin and
Number of the strains
A
0
Class
leucine arylamidase (III: Supplementary
table S1). In addition, all were positive
for acid phosphatase, naphthol-AS-BIphosphohydrolase, phosphodiesterase,
esterase, lipase and lipase esterase. The
enzyme reactions varied from weak to
strong. Most of the 13 strains were also
positive for phosphomonoesterase and
cysteine and valine arylamidases. The
cells grew heterotrophically under aerobic
conditions. All strains utilized Tween 40 as
a carbon source but only few other carbon
sources were utilized (III: Supplementary
table S2). The results indicated that the
strains had a high potential to mineralize
organic phosphorus compounds and
that they utilized amino acid residues.
In contrast, they had only limited
potential in using common environmental
macromolecules for carbon and sulphur
sources.
The cells of the strains were Gramnegative, rod-shaped (0.5–0.7×1.3–5.0
Actinobacteria
Flavobacteria
Sphingobacteria
Deinococci
Bacilli
Alphaproteobacteria
Betaproteobacteria
Gammaproteobacteria
40
80
120
160
200
240
280
320
360
5%
7%
3%
02%
3%
16%
9%
57%
TOX
CYA
R2A
Z8
BA
SAA
Figure 1: Taxonomic distribution of the isolated heterotrophic bacterial strains in total and in
relation to the used isolation media.
A: at the class level. The percentage of the strains representing each class, of the total number of
the isolated strains, is shown together with the class bars.
B: at the genus level. TOX: medium with demethyl variants of microcystins. CYA: medium
with frozen and thawed culture of non-microcystin-producing Anabaena. R2A: Reasoner’s 2A
medium. Z8: nutrient poor medium. BA: blood agar. SAA: starch ampicillin agar.
22
Results
Number of the strains
B
1
2
Chryseobacterium
Flavobacterium
Riemerella
Assigned at family level
3
Arcicella
Chitinophaga
Flectobacillus
Pedobacter
Spirosoma
4
Deinococcus
5
Bacillus
Caryophanon
Lactococcus
Paenibacillus
Staphylococcus
6
Azospirillum
Blastomonas
Bosea
Brevundimonas
Caulobacter
Methylobacterium
Novosphingobium
Paracoccus
Phyllobacterium
Porphyrobacter
Sphingobium
Sphingomonas
Sphingopyxis
Assigned at family level
Assigned at class level
7
Aquabacterium
Aquitalea
Burkholderia
Chitinimonas
Chromobacterium
Duganella
Herbaspirillum
Iodobacter
Janthinobacterium
Pelomonas
Rhodoferax
Roseateles
Vogesella
Assigned at family level
Assigned at class level
8
Acinetobacter
Aeromonas
Erwinia
Pseudomonas
Rheinheimera
Serratia
Stenotrophomonas
Vibrio
Assigned at family level
10
20
30 33 121 282
1 Actinobacteria 2 Flavobacteria 3 Sphingobacteria 4 Deinococci 5 Bacilli
6 Alphaproteobacteria 7 Betaproteobacteria 8 Gammaproteobacteria
Class (1–8) and genus
0
Arthrobacter
Kocuria
Microbacterium
Micrococcus
Mycobacterium
Nocardia
Nocardioides
Rhodococcus
Streptomyces
Assigned at family level
TOX
TOX
CYA
CYA
R2A
SAA
R2A Z8 Z8 BA BA
SAA
23
Results
μm) and motile by a single polar flagellum
(III). They formed greyish colonies on the
relatively oligotrophic R2A medium, but
did not grow on rich growth media such
as trypticase soy agar (TSA; III). The
diameter of the colonies on the R2A was
≤1 mm after incubation for 65 h at 20±2
ºC. The cells grew well at 20–30 ºC but
not at 37 ºC. The main fatty acids were
C16:0, C16:1ω7c; and ω7c, ω9c and ω12t
forms of C18:1 (III: Supplementary table
S3). The guanine-cytosine content of the
genomic DNA was 66.1–68.0 mol% (III:
Supplementary table S2). On the basis
of these characteristics a new genus and
species Paucibacter toxinivorans was
described, with strain 2C20 as the type
strain.
4.1.3 Virulence potential of the
heterotrophic bacteria associated with
cyanobacteria
Haemolytic strains representing 11 genera
were isolated by choosing haemolytic
colonies from the blood agar (Fig. 2; I:
supplementary data). The most dominant
genera among the haemolytic strains were
Aeromonas spp. (69%), Acinetobacter spp.
(8%), Vibrio spp. (8%) and Pseudomonas
spp. (5%). The haemolytic characteristic
of the strains implied their potential
virulence.
Due to the high proportion of the
haemolytic Aeromonas spp. strains this
genus was targeted for a closer study. The
majority (90%) of the 176 Aeromonas
strains isolated from the fresh and brackish
Pseudomonas; 6; 5%
Rheinheimera; 2; 2%
Vibrio; 11; 8%
Vibrionaceae; 1; 1%
Flavobacteriaceae; 1; 1%
Aeromonas; 92; 69%
Bacillus; 2; 2%
Staphylococcus; 1; 1%
Brevundimonas; 1; 1%
Aquitalea; 1; 1%
Burkholderia; 1; 1%
Chromobacterium; 4; 3%
Acinetobacter; 10; 8%
Bacteroidetes:
Proteobacteria:
Flavobacteria Firmicutes:
Alphaproteobacteria
Betaproteobacteria
Bacilli
Gammaproteobacteria
Figure 2: Taxonomic distribution of the isolated haemolytic bacterial strains. The number of the
strains assigned at each species or family level and their percentage of the haemolytic strains
(n=133) are shown together with their taxon.
24
Results
water samples that were associated with
various adverse health effects in humans
contained at least one out of the six
screened Aeromonas virulence gene types,
based on the PCR analysis (II: Fig. 2). The
most common virulence gene type was the
cytotoxic enterotoxin and haemolytic gene
products encoding act (act/aerA/hlyA) that
was present in 77% of the strains studied
(II: Fig. 2). The flagellin subunits encoding
fla (flaA/flaB), elastase encoding ahyB and
lipase encoding lip (lip/pla/lipH3/alp-1)
gene types were present in 30–37% of
the strains. The counts of the presumptive
Aeromonas colonies measured on the
starch ampicillin agar medium from the
water samples varied between 5−4.5×104
colony forming units (CFU) ml -1 with
a median of 68 CFU ml-1 (II). The most
common health effects reported by
the affected persons were fever and
gastrointestinal disturbances (II: Fig. 1).
The common occurrence of the virulence
genes among the studied Aeromonas
strains indicated a virulence potential of
the strains and suggested that they may be
the source of some of the reported adverse
health effects.
The Aeromonas strains that were
screened for the virulence genes formed
three genogroups (II: Table 3). The
cytotonic enterotoxin encoding ast
gene was strongly associated with the
genogroup III that contained the tentative
A. hydrophila and A. media strains,
identified on the basis of their partial 16S
rRNA gene sequences (II: Fig. 3). The lip,
fla, ahyB and the cytotonic enterotoxin
encoding alt genes were associated
most strongly with the genogroup II that
contained the tentative A. salmonicida,
A. popoffii and A. sobria strains. The
associations were studied using canonical
discriminant analyses. The most abundant
virulence gene type, act, was detected
in strains of all the three genogroups,
including also 73% of the strains in
genogroup I that contained the tentative A.
veronii and A. jandaei strains (II: Fig. 2).
The results suggested a higher virulence
potential of the strains in genogroups II
and III compared to those of genogroup I.
The water samples, from which the
screened Aeromonas strains were isolated,
were also analysed for cyanobacterial
hepatotoxins, neurotoxins and bacterial
endotoxins (II). The concentrations of the
hepatotoxins microcystins and nodularin
varied from below the detection limit of
0.1, to 12.3 μg l-1 in the majority (97%)
of the analysed samples (II: Table 2). The
median of the concentrations was below
the detection limit of 0.1 μg l-1. Of the
neurotoxins, low concentrations (1.9–10.3
μg l -1) of anatoxin-a, homoanatoxin-a
or their degradation products were
detected in 4 of the 22 analysed samples
and acetylcholinesterase inhibition that
suggested the presence of anatoxin-a(S)
in 1 of the 31 analysed samples. The
presence of saxitoxins was not detected
in the 27 samples analysed. The measured
concentrations of bacterial endotoxin
varied from 10 to 300 endotoxin units
(EU) ml-1 in the majority (89%) of the
samples, with the median of 58 EU
ml -1. The estimated endotoxin aerosol
concentrations of the majority (89%) of
the samples varied between 0.3–60.4
EU m -3 (II). Based on the resuts, the
analysed toxins were either present in low
concentrations or undetectable from the
samples.
4.1.4 Effects of the heterotrophic
bacteria on the growth of
cyanobacteria
The majority (60%) of the 183
heterotrophic bacterial strains analysed
either enhanced or inhibited the growth of
the microcystins-producing Microcystis
PCC 7941 or the non-microcystin25
Results
producing Anabaena PCC 7122
strain when they were incubated with
cyanobacterial cells on agar plates (Fig. 3;
I: Table 3). The growth of one or both of
the cyanobacterial strains was enhanced by
48% of the heterotrophic bacterial strains
and inhibited by 10% of the strains. Two
of the strains (1%) enhanced the growth
of one of the test cyanobacterial strains
and inhibited the growth of the other.
Most notably, the cyanobacterial growth
was enhanced by over 50% of the tested
Alphaproteobacteria, Betaproteobacteria
and Actinobacteria strains. Overall, the
isolated strains representing all eight
bacterial classes and 30 different genera
enhanced or inhibited the cyanobacterial
growth. The results implied that the ability
to affect cyanobacterial growth is common
among the cyanobacteria associated
heterotrophic bacteria.
A
Number of the strains
0
Actinobacteria
Flavobacteria
Class
4.2 Selectivity of the used growth
media
The strains isolated using the TOX
medium that contained demethyl variants
of microcystins, represented all 8 classes
and 51% of the total 57 genera that were
identified among the bacterial strains
isolated in this study (Fig. 1, Appendix
1; I: Fig 2). The strains from the CYA
medium, which contained frozen and
thawed culture of non-microcystinproducing Anabaena and compounds for
allowing the isolation of slow growing
bacteria, represented 7 classes and 47%
of the genera. Those growth media had
been formulated to yield putative utilisers
of cyanobacterial biomass and toxins. The
strains from the R2A medium, chosen to
yield high numbers of different bacteria,
represented 6 classes and 46% of the
genera. The strains from the nutrient poor
Sphingobacteria
Deinococci
5
10
15
54%
29%
20
25
30
35
40
45
50
55
17%
22%
59%
35% 25%
40%
19%
100%
Bacilli 25% 50% 25%
Alphaproteobacteria
Betaproteobacteria
Gammaproteobacteria
35%
36%
60%
58%
4% 2%
3%3%
55% 45%
No effect
Enhanced
Inhibited
Enhanced/inhibited
Figure 3: Effect of the tested heterotrophic bacterial strains on the growth of the microcystinsproducing Microcystis PCC 7941 and non-microcystin-producing Anabaena PCC 7122 strains.
A: at the class level. The percentages of strains with specific effect on cyanobacterial growth, of
the total number of tested strains within each class, are shown together with the class bars.
B: at the genus level. The strains identified only at family level are underlined. No effect: the
strain did not affect the cyanobacterial growth. Enhanced: the strain enhanced the growth of one
or both of the cyanobacterial strains. Inhibited: the strain inhibited the growth of one or both
of the cyanobacterial strains. Enhanced/inhibited: the strain enhanced the growth of one of the
cyanobacterial strain and inhibited the growth of the other.
26
Results
B
Number of the strains
1
2
Chryseobacterium
Flavobacterium
Riemerella
3
Arcicella
Chitinophaga
Flectobacillus
Pedobacter
Spirosoma
4
Deinococcus
5
Bacillus
Staphylococcus
6
Azospirillum
Bosea
Brevundimonas
Caulobacter
Methylobacterium
Novosphingobium
Paracoccus
Sphingobium
Sphingomonas
Sphingopyxis
Acetobacteraceae
7
Duganella
Herbaspirillum
Iodobacter
Janthinobacterium
Pelomonas
Rhodoferax
Roseateles
Vogesella
Incertae sedis 5
Oxalobacteraceae
8
Acinetobacter
Aeromonas
Pseudomonas
Rheinheimera
Stenotrophomonas
No effect
5
10
15
20
25
30
1 Actinobacteria 2 Flavobacteria 3 Sphingobacteria 4 Deinococci 5 Bacilli
6 Alphaproteobacteria 7 Betaproteobacteria 8 Gammaproteobacteria
Class (1–8) and genus or family
0
Arthrobacter
Kocuria
Microbacterium
Micrococcus
Nocardioides
Rhodococcus
Streptomyces
Microbacteriaceae
Enhanced
Inhibited
Enhanced/inhibited
27
Results
Z8 medium represented four classes and
11% of the genera. The strains from the
blood agar (BA) medium, from which
haemolytic colonies were primarily
chosen, represented 6 classes and 28%
of the genera. The Z8 media was chosen
for yielding the most oligotrophic bacteria
and the BA medium chosen for yielding
putative pathogenic bacteria.
Altogether, the strains isolated using
TOX, CYA and R2A media represented a
majority (84%) of the 57 isolated genera
(Fig. 1, Appendix 1; I: Fig 2). However,
genera such as Rhodococcus, Aeromonas
and Pseudomonas were represented only
or mostly by strains isolated using the
BA or Z8 media. Based on these results,
combination of TOX, CYA and R2A media
was suitable for isolating a high variety of
heterotrophic bacteria co-occurring with
cyanobacteria but the BA and Z8 media
provided a notable addition of putative
pathogens and oligotrophic bacteria to the
total number of isolated strains.
Of the presumptive Aeromonas
strains obtained from the starch
ampicillin agar (SAA) medium, 95%
were verified as Aeromonas, whereas
the remaining 5% were assigned to the
genera Chryseobacterium and Erwinia,
or the family Enterobacteriaceae (Fig. 1,
Appendix 1; II: Table 3). The SAA medium
had been chosen as a selective growth
medium for Aeromonas spp. isolation. The
results indicated that the SAA medium was
selective for the isolation of Aeromonas
even from samples as complex as the
cyanobacterial water blooms.
4.3 Drinking water treatment
efficiency
The efficiency of the drinking water
treatment process during cyanobacterial
mass occurrence in the source water was
assessed at an operating water treatment
28
plant (IV). The assessed factors were the
removal of planktonic cells, cyanobacterial
hepatotoxins, cultivable heterotrophic
bacteria and endotoxins. The water
treatment process consisted of coagulation
with aluminium salts, clarification by
sedimentation, sand filtration, ozonation,
slow sand filtration and chlorination. In
addition, the taxonomic distribution of the
heterotrophic bacterial genera originating
from treated drinking water (included
in 4.1.1) of the above mentioned water
treatment plant and of six other plants was
assessed (I, IV).
4.3.1 Removal of toxins by the water
treatment
The microcystin concentrations in the
treated drinking water varied from <0.02
to 0.03μg l-1, based on the enzyme-linked
immunosorbent assay (ELISA) and protein
phosphatase inhibition analyses (IV: Fig 2).
The concentrations of microcystins in the
incoming water varied from <0.02 up to 10
μg l-1. The maximum concentration in the
treated water was detected during ozonator
malfunction. During normal operation, the
coagulation, clarification, sand filtration
and ozonation steps usually reduced the
microcystin concentrations below the
detection limit of 0.02 μg l-1, indicating
efficient removal of the microcystins
from water in the first phases of the water
treatment also during cyanobacterial mass
occurrences.
The endotoxin activities in the treated
drinking water were low, 4–38 EU ml-1,
based on the kinetic chromogenic Limulus
amoebocyte lysate analysis (IV: Table 3).
In contrast, endotoxin concentrations of
up to 3,300 EU ml-1 were measured in the
source water. Coagulation, clarification
and sand filtration reduced the endotoxins
by 0.72–2.03 log10 units, and ozonation by
0.04–0.19 log10 units. The efficiency of the
Results
slow sand filtration varied from ineffective
to a maximum reduction of 1.35 log10 units
and the chlorination step was ineffective
against endotoxins. However, assessment
of the efficiency of the last treatment
steps was hindered by the low endotoxin
concentrations encountered after the
preceding water treatment steps. The
results indicated that water treatment,
especially the coagulation-sand filtration
steps, removed endotoxins efficiently.
Signs of neurotoxic agent were
detected by the Artemia salina
bioassay, which was used to assess
the overall toxicity, in samples taken
from cyanobacterial water blooms that
occurred at or upwards of the water
intake of the waterworks (IV: Table 1).
The neurotoxin remained unidentified,
even though the samples were tested
for anatoxin-a, homoanatoxin-a and
their epoxy and dihydro degradation
products by high-performance liquid
chromatography (HPLC), anatoxina(S) by acetylcholinesterase inhibition
assay, and saxitoxins by Jellett rapid
paralytic shellfish poisons test (IV). The
most dominant cyanobacterial species in
seven out of nine samples, and second
most dominant in one, of the neurotoxic
bloom samples was Anabaena cf. smithii,
implicating it as the most probable origin
of the neurotoxic agent.
4.3.2 Removal of planktonic cells by the
water treatment
Large phytoplankton, belonging mainly
to the size class of >65 μm3 were rarely
(1–5 times out of 27 sampling dates)
detected in the treated drinking water
based on the microscopic analyses, with
the exception of Oscillatoriales spp.
filaments (IV: Table 2). The maximum
count of cells, colonies or filaments (100
μm) of large phytoplankton detected in
the treated water was 2.80×104 counting
units l-1 and the maximum wet weight
14.13 μg l-1. In contrast, phytoplankton
wet weight concentrations of >1.0×103
μg l-1 were detected on several occasions
from the incoming source water (IV: Fig.
1). This indicated that the water treatment
process was efficient at removing large
phytoplankton.
Picoplankton (size class ≤2 μm 3,
including possible bacterial cells) and
monad cells (size class 6–65 μm 3 )
were detected in the treated drinking
water 25 to 22 times out of 27 sampling
dates, respectively (IV: Table 2). The
picoplankton cells were present up to
1.73×106 counting units l-1 (3.46 μg l-1) and
the monads up to 2.27×105 counting units
l-1 (5.12 μg l-1). The results indicate that
the picoplankton and monad cells could
pass the treatment system quite regularly.
Oscillatoriales spp. filaments (size
class 314 μm3) were present in low (up to
2.80×104 counting units l-1) concentrations
in the treated water on 20 occasions out
of 27 sampling dates (IV: Table 2). The
filaments were not detected in the source
water, which implied that they originated
inside the water treatment facilities.
The heterotrophic bacterial counts
measured in the treated drinking water
were mostly low, usually less than 1 CFU
ml-1, based on the colony counts on R2A
plates (IV). The chlorination reduced
the cultivable heterotrophic bacterial
counts by >3 log10 units, coagulation-sand
filtration by 0.6–1.0 log10 units and the
slow sand filtration step by 0.5–1.0 log10
units. In contrast the ozonation step was
ineffective. The total range of bacterial
removal was 2.0–5.0 log10 units, varying
generally between 4.0–5.0 log10 units. The
results indicated efficient removal of the
cultivable heterotrophic bacteria during
the water treatment process.
29
Results
4.3.3 Taxonomy of the heterotrophic
bacteria isolated from drinking water
The majority (54%) of the 61 bacterial
strains isolated from the treated drinking
water, belonged to the Alphaproteobacteria
(Fig. 4, Appendix 1; I; IV). Strains of this
class assigned to the genera Sphingomonas
and Brevundimonas were isolated from the
treated drinking water of five of the seven
drinking water treatment plants studied,
regardless of the used treatment system
(I, IV). In total, strains from 19 different
genera of the classes Actinobacteria (3
genera), Flavobacteria (2 genera), Bacilli
(3 genera), Alpha- (7 genera), Beta- (2
genera) and Gammaproteobacteria (2
genera) were isolated from the treated
drinking waters (Fig. 4, Appendix 1; I:
Supplementary data; IV). In addition,
two strains could only be assigned at
the family level as Sphingomonadaceae
and two others as Oxalobacteraceae. All
but four of the genera isolated from the
treated drinking waters were also isolated
from the freshwater samples (Fig. 4, I:
Supplementary data). This implied that the
freshwater bacteria are the most probable
source of the cultivable bacteria passing
through water treatment processes.
Micrococcus: 1/ES, 1/RA
Mycobacterium: 1/ES
Nocardia: 1/KK
Pseudomonas: 3/RN, 4/SL, 1/VA
Chryseobacterium: 1/VA
Flavobacterium: 2/KK
Acinetobacter: 1/SL
Bacillus: 2/ES, 1/RN
Paenibacillus: 1/ES
Staphylococcus: 1/SL
Oxalobacteraceae:
1/KK, 1/RN
Herbaspirillum:
1/KK, 2/RN
Blastomonas: 2/RN
Duganella: 2/KK
Bosea:
1/KK, 1/RA, 1/RN
Sphingomonadaceae:
1/RA, 1/RN
Sphingopyxis: 1/RA
Sphingomonas:
4/HV, 2/RA, 1/RN,
2/SL, 2/VA
Brevundimonas:
1/HV, 1/KK, 1/RA,
3/SL, 1/VA
Methylobacterium: 4/ES
Phyllobacterium: 2/HV, 1/VA
Actinobacteria:
Actinobacteria Bacteroidetes:
Proteobacteria:
Alphaproteobacteria
Flavobacteria Firmicutes:
Betaproteobacteria
Bacilli
Gammaproteobacteria
Figure 4: Taxonomic distribution of the heterotrophic bacterial strains (n=61) isolated from
treated drinking water. The number of the strains and the treatment plant (ES, HV, KK, RA, RN,
SL or VA) they originated from are given together with the taxon. The four genera which in this
study were only isolated from treated drinking water are underlined.
30
Discussion
5. DISCUSSION
5.1 Characteristics of the isolated
heterotrophic bacteria
The heterotrophic bacterial strains isolated
from natural water or treated drinking
water with increased cyanobacterial
occurrence in the water bodies or in the
source water for drinking water treatment
represented a wide variety of bacterial
taxa (I, II, IV). They also included several
strains of potential new bacterial taxa from
species to order levels. The majority of the
isolated bacterial strains isolated in this
study were assigned to the Alpha-, Betaand Gammaproteobacteria. Bacteria of
these classes have typically been found
in freshwater, and in association with
cyanobacterial blooms, in studies that
used cultivation-independent molecular
methods (Zwart et al. 2002, Eiler and
Bertilsson 2004, Kolmonen et al. 2004).
In contrast, bacteria of taxa such as the
Verrucomicrobia that have been detected
previously by cultivation-independent
methods (e.g. Eiler and Bertilsson 2004,
Kolmonen et al. 2004), were not detected
among the strains isolated in this study.
Cultivation methods can be laborious and
the difficulties in finding suitable growth
conditions for any particular species
narrow the bacterial taxons detected using
these methods (Hugenholtz et al. 1998,
Zinder and Salyers 2001). Nevertheless,
the studies with isolated strains provide
useful reference points of bacteria with
known characteristics also for the analyses
done by the cultivation-independent
molecular methods (Zinder and Salyers
2001). Therefore, both cultivation and
cultivation-independent molecular
methods are needed to improve our
knowledge on the bacterial communities
and their roles in the environment.
Among the isolated heterotrophic
bacterial strains were more than 100
haemolytic strains, which represented
11 different genera, predominantly the
genus Aeromonas (I). Their taxonomic
assignment and haemolytic properties
implied virulence potential. Furthermore,
the majority (90%) of the tested
Aeromonas strains contained at least one
of the six Aeromonas virulence gene types
screened for in this study, suggesting
a virulence capacity of the strains (II).
The most common of the detected gene
types was the cytotoxic enterotoxin and
haemolytic gene products encoding act
(act/aerA/hlyA). The tested strains were
isolated from aquatic samples that were
associated with adverse health effects
in humans, most commonly fever or
gastrointestinal disturbances, surmised
to be caused by cyanobacterial water
blooms. However, health effects such as
fever and gastrointestinal disturbances are
also associated with Aeromonas (Janda
and Abbott 1998, Stewart et al. 2006b).
The cytotoxic enterotoxin encoded by
the act gene induces an inflammatory
response in host cells (Galindo et al.
2006) that can contribute to systemic
effects such as fever. The presence of
act has also been connected to bloody
diarrhoea, and in a mouse test, a mutant
Aeromonas with an act deletion induced
64% lower fluid secretion than the wild
type Aeromonas (Albert et al. 2000,
Sha et al. 2002). The haemolytic toxins
encoding aerA and hlyA genes have also
been shown to contribute to the virulence
of A. hydrophila in studies using aerA
and hlyA inactivated mutants and mice
tests (Wong et al. 1998). The common
presence of act and the other virulence
31
Discussion
genes suggests that the Aeromonas strains
may be the source of the detected human
health effects, especially those of fever
and gastrointestinal disturbances.
In the toxin analysis of the water
samples from which the Aeromonas strains
screened for the virulence genes originated
from, the bacterial endotoxins were
detected in relatively low concentrations
in the majority of the samples (II).
The cyanobacterial hepatotoxins and
neurotoxins concentrations of the majority
of the samples were below the detection
limits. Little information about the effects
of endotoxin exposure by ingestion is
available. However, for aerosol exposure
a quideline of 10 ng m -3 (~100 EU
m -3) for no observed effects of airway
inflammations and 100 ng m-3 (~1000 EU
m-3) for no observed systemic effects has
been proposed (Anderson et al. 2002 and
2007). The estimated endotoxin aerosol
concentrations (<1–60 EU m -3) of a
majority of the samples is this study were
below 100 EU m-3 guideline. Hepatotoxin
concentrations of up to 10 μg l -1 of
microcystins are considered to present a
relatively low probability of adverse health
effects (Falconer et al. 1999). Furthermore,
in a study by Fawell et al. (1999) using
animal models the no observed adverse
effect level dose of orally given anatoxin-a
was 98 μg kg-1 body weight. Therefore,
the detected toxins by themselves are
unlikely to be the cause of the reported
gastrointestinal disturbances or fever.
In addition to Aeromonas, haemolytic
strains of various other genera such as
Vibrio, Pseudomonas and Acinetobacter
were isolated (I). These genera are
known to contain both human and animal
pathogens (Farmer et al. 2005, Palleroni
2005, Peleg et al. 2008). For example,
Vibrio cholerae is a well known pathogen
for cholera disease, which is caused by
32
V. cholerae of the serogroups O1 and
O139 (Faruque et al.1998, Farmer et al.
2005). In addition, adverse health effects
causing pathogenic strains exist also in
other Vibrio species and among noncholera V. cholerae strains (Thompson et
al. 2004, Lukinmaa et al. 2006). In a study
by Eiler et al. (2006a) members of this
genus were regularly detected in the Baltic
Sea, including also populations containing
mostly V. cholerae. Their study indicated
that vibrios may potentially be endemic
pathogens of the cold and brackish waters
of the Baltic Sea (Eiler et al. 2006a), from
which the Vibrio strains of the present
study also originated (I). The association
with cyanobacterial cells has been shown
to prolong cultivable and viable state of
vibrios, and cyanobacterial blooms have
been proposed to function as a natural
reservoir for Vibrio (Islam et al. 1999,
2002 and 2004). Therefore, the presence
of bacteria such as Vibrio further supports
the importance of assessing the health
risks posed by the cyanobacterial blooms
as a whole.
In contrast to the putative pathogens,
the 13 previously isolated cyanobacterial
hepatotoxins degrading Betaproteobacteria
that were further characterised in this study
may reduce the health risks associated
with cyanobacterial water blooms. These
Betaproteobacteria strains that could
not be assigned to any described genus
or species previously, were described
as a new genus and species Paucibacter
toxinivorans (III). Tested Paucibacter
toxinivorans strains degraded nodularin,
with a considerably higher degradation rate
than that of the microcystins. These strains
were isolated by Lahti et al. (1998) from
sediments of Finnish lakes with frequent
cyanobacterial occurrence. The majority
of the previously isolated bacterial strains
that are able to degrade cyanobacterial
Discussion
hepatotoxins have belonged to the family
Sphingomonadaceae, most commonly to
the genus Sphingomonas (e.g. Jones et al.
1994, Park et al. 2001, Saito et al. 2003,
Saitou et al. 2003, Maryama et al. 2006,
Ho et al. 2007). Only few isolated bacterial
strains capable of degrading cyanobacterial
microcystins have also been shown to be
able to degrade nodularin. These include
two Paucibacter toxinivorans strains tested
previously for nodularin degradation,
and an unidentified Sphingomonas strain
isolated by Lahti et al. (1998) as well
as two Sphingomonas strains, the 7CY
isolated by Ishii et al. (2004) and the B9
(Imanishi et al. 2005) isolated by Harada
et al. (2004). The Paucibacter toxinivorans
strains widened the taxonomic distribution
of isolated bacteria known to degrade
cyanobacterial hepatotoxins, especially
nodularin and showed that the ability to
degrade the hepatotoxins is not restricted
to Sphingomonadaceae.
The majority (60%) of the isolated
heterotrophic bacterial strains that
were tested for their ability to affect
cyanobacterial growth either enhanced or
inhibited the growth (I). They represented
all eight bacterial classes embodied
among the strains isolated in this study
and 30 different genera in total. The fact
that the majority (81%, 29 genera) of the
strains that affected the cyanobacterial
growth enhanced the growth indicated
a mutualistic interaction between these
bacteria and the cyanobacteria. Previously
known cyanobacterial growth affecting
bacteria have represented relatively few
taxa such as Alcaligenes, Streptomyces,
Cytophaga, Pseudomonas and Shewanella
(Yamamoto et al. 1998, Dakhama et al.
1993, Manage et al. 2000, Rashidan and
Bird 2001, Salomon et al. 2003). The exact
mechanisms, by which the bacteria enhance
or inhibit the cyanobacterial growth is
often unknown. In many cases the studied
effect has been growth inhibiting, and lytic
bacteria have been proposed as a short
term means for reducing problems caused
by cyanobacterial water blooms (Rashidan
and Bird 2001). However, long term
control would require better understanding
also on the effects supporting the
cyanobacterial water bloom formation.
The strains isolated in this study provide a
diverse group of bacteria for studying the
mechanisms behind cyanobacterial growth
inhibition and enhancement by individual
bacterial strains or strain combinations.
The isolated strains of genera
such as Streptomyces, Flavobacterium,
Sphingomonas and Acinetobacter also
provide a wide range of putative producers
of bioactive compounds or degraders
of recalcitrant organic compounds (I).
Members of Actinobacteria, most notably
bacteria of the genus Streptomyces, are
known to produce a wide variety of
bioactive compounds, including over half
of the compounds listed in the Antibiotic
Literature Database (Lazzarini et al.
2000). Members of the Flavobacterium
and Sphingomonas genera are known
to degrade various kinds of complex
organic or synthetic compounds (Balkwill
et al. 2006, Bernardet and Bowman
2006) such as crude oil or the endocrinedisruptive phthalate esters (Baraniecki
et al. 2002, Rahman et al. 2002, Liang
et al. 2008). Production of bioactive
compounds and versatile degradation of
organic compounds could explain the
effects that many of these strains have on
cyanobacterial growth and the wide range
of bacteria associated with cyanobacteria.
However, these potential biosynthetic or
degradation abilities were not investigated
in this study.
33
Discussion
5.2 Drinking water treatment
efficiency
The drinking water treatment steps
of coagulation, clarification and sand
filtration were the most efficient as a
whole in reducing both hepatotoxin
and endotoxin concentrations as well
as heterotrophic bacterial counts (IV).
The results were in accordance with
previous studies by Lepistö et al. (1994)
and Rapala et al. (2002b). These steps
are used mainly to remove particulate
matter, such as microbial cells and with
them their endotoxins (WHO 2008b,
LeChevallier and Au 2004d). As the
majority of the hepatotoxins produced by
the cyanobacteria remain inside the cells
until the cells die, the coagulation-filtration
steps also reduce the hepatotoxins as long
as the cyanobacterial cells remain intact
(Hitzfeld et al. 2000, Svrcek and Smith
2004). The hepatotoxin concentrations may
be further reduced due to biodegradation
by microbial communities within the sand
filters as was shown in the study of Ho
et al. (2006 and 2007). The importance
of these combined treatment steps in
effective removal of cells and their toxins
was supported by the results of this study.
The efficiencies of the ozonation,
slow sand filtration and chlorination steps
on removal of microcystins, endotoxins
and heterotrophic bacteria varied
depending on the measured agent (IV).
The most variable results were obtained
with the ozonation step, which was
efficient in the removal of microcystins.
In contrast, it only slightly reduced the
endotoxin amounts and was inefficient
against the heterotrophic bacteria. The
inefficiency of the ozonation step in the
removal of heterotrophic bacteria and
endotoxins may be partly explained
by the prevalence of bacteria such as
the Sphingomonas and Brevundimonas
34
among the strains isolated from the treated
drinking water (I, IV). Bacteria of these
genera are known carotenoid producers
(Masetto et al. 2001, Asker et al. 2007).
The production of pigments such as
carotenoids and flavonoids may protect
the bacteria from the harmful effects of the
ozonation (LeChevallier and Au 2004a).
Therefore the pigment production may
allow bacteria, such as Sphingomonas and
Brevundimonas, pass through the water
treatment system in a viable state.
Picoplankton and monad cells
regularly passed through the water
treatment process (IV). In contrast,
the larger phytoplankton particles
rarely passed through the treatment
process, implying particle size to have a
determining role in plankton removal.
Attachment to particles protects bacteria
from disinfection processes such as
chlorination (LeChevallier and Au
2004a). The oxidation of the cell matter
also consumes ozone (Svrcek and Smith
2004). Therefore, the presence of the
phytoplankton cells may help viable
heterotrophic bacteria to pass through the
water treatment process and also might
hinder the destruction of the bacteria
by secondary disinfection during water
distribution.
The Oscillatoriales filaments detected
in the drinking water were not detected in
the untreated source water, which indicates
that they originated from inside the water
treatment facilities (IV). The sand filters,
especially the slow sand filters have
biological layer as a functional component
within which photosynthetic organisms
may function as important carbon inputs
(Campos et al. 2002, LeChevallier and Au
2004d, WHO 2008b). Therefore, the most
probable source of the Oscillatoriales
filaments was the slow sand filter.
Consequently, the Oscillatoriales
Discussion
concentrations in the treated water might
be reduced by covering the slow sand filter
to prevent photoautotrophic functions.
The heterotrophic bacterial strains
isolated from treated drinking water
of the seven drinking water treatment
plants with differring water treatment
procedures, were dominated by bacteria
of the Alphaproteobacteria class (I,
IV). Genera such as Mycobacterium,
Flavobacterium and Pseudomonas, of
the classes Actinobacteria, Flavobacteria
and Gammaproteobacteria, respectively,
were also present. Bacteria of the
Alphaproteobacteria class were also
found to predominate in the treated
distribution water in a distribution system
simulator study by Williams et al. (2004).
In addition, they were one of the major
groups of bacteria isolated from a water
distribution system in a study by Tokajian
et al. (2005). These findings indicate the
ability of Alphaproteobacteria to survive in
water supply systems. Microbes, including
bacterial genera such as Pseudomonas and
Mycobacterium, produce biofilms on the
surfaces of water distribution systems,
which can lower the obtained drinking
water quality and cause corrosion of the
distribution system (Percival and Walker
1999, Norton and LeChervallier 2000,
LeChevallier and Au 2004c, Payment and
Robertson 2004, Berry et al. 2006). They
can also protect other bacteria, including
pathogenic strains, from removal by
disinfectants and water flows. Therefore,
detected viable heterotrophic bacteria may
cause problems within water distribution
systems.
35
Conclusions
6. CONCLUSIONS
This study provided information on
the heterotrophic bacterial community
associated with the cyanobacterial water
blooms and on the capability of modern
drinking water treatment processes to
cope with the extra burden caused by such
water blooms.
The removal of phytoplankton,
cyanobacterial hepatotoxins, endotoxins
and heterotrophic bacteria by the water
treatment was for the most part highly
efficient. However, the cells passing
through the treatment process may in
the long term cause problems such as
microbial proliferation in biofilms,
corrosion of water conducting pipes, a
deterioration in the water distribution
system and a reduction in the drinking
water quality. The water treatment steps
varied in their efficiency in reducing
the detected toxins and organisms. This
supports the importance of combining
multiple treatment steps to ensure overall
good quality of drinking water.
The heterotrophic bacterial strains
isolated from treated drinking water
predominantly represented genera
that were also isolated from untreated
freshwater, which implies the freshwater
bodies as being the original source.
Overall, the isolated bacterial strains
were taxonomically widely distributed,
which showed that the cyanobacterial
water blooms are associated with a diverse
community of cultivable heterotrophic
bacteria. The majority of the tested strains
either enhanced or inhibited the growth
of the cyanobacterial strains Microcystis
PCC 7941 and Anabaena PCC 7122. In
most cases they enhanced the growth of
cyanobacteria, which implies mutualistic
interactions between the heterotrophic
36
bacteria and cyanobacteria. The common
ability to affect cyanobacterial growth
indicates that the heterotrophic bacteria
may have a role in the formation and
decline of the cyanobacterial water
blooms.
The taxonomic assignment and
haemolytic features of many of the isolates
indicated that the blooms harbour a wide
variety of putative pathogenic bacteria.
The most prominent group of such bacteria
were the Aeromonas strains, which were
isolated from both fresh and brackish
water samples. The majority of the tested
Aeromonas strains included one or more
of the screened Aeromonas virulence
gene types, most commonly that of the
cytotoxic enterotoxin and haemolytic
products encoding act. The prevalence of
the screened virulence genes within the
Aeromonas strains further implies that
they have virulence potential for both
humans and animals. Therefore, they may
pose health risks and economic losses to
the recreational and potable use of water.
The unidentified Betaproteobacteria
strains previously isolated from lakes
with frequently occurring cyanobacterial
water blooms degraded the cyanobacterial
hepatotoxins, microcystins and nodularin.
Based on their genetic and phenotypic
characteristics they were described as
a new genus and species Paucibacter
toxinivorans. These strains widen
the spectrum of known degraders of
cyanobacterial hepatotoxins, especially
nodularins, and thus give support for the
role of microbial biodegradation in the
removal of the chemically stable toxins
from the environment. In addition, the
isolated bacterial strains representing
genera such as Sphingomonas and
Conclusions
Flavobacterium may include new
degraders of cyanobacterial hepatotoxins
and other complex organic compounds.
The heterotrophic bacteria of this
study offer a rich source of strains for
examination of both mutual and biased
interactions between cyanobacteria and
the heterotrophic bacteria associated
with them. The putative pathogenicity of
many of the strains support the need to
widen the health risk assessment of the
cyanobacterial water blooms to cover also
the heterotrophic bacterial community
associated with them. However, the
cyanobacterial water blooms also seem
to harbour a wide variety of putative
producers of bioactive compounds or
degraders of recalcitrant compounds.
Therefore, both cyanobacteria and their
heterotrophic associates should be taken
into consideration, when studying the
growth dynamics and risks as well as
opportunities posed by the cyanobacterial
water blooms.
37
Acknowledgements
7. ACKNOWLEDGEMENTS
This work was carried out at the Finnish Environment Institute and the Department
of Applied Chemistry and Microbiology, Division of Microbiology. The Academy of
Finland (Microbes and Man (MICMAN) Research Programme, grant 201356 awarded
to Jarkko Rapala and the Centres of Excellence Programmes, grants 53305 and 118637
awarded to Kaarina Sivonen), the Finnish Environment Institute and the University of
Helsinki are gratefully thanked for their financial support.
I would like to express my special gratitude to my supervisors, Docent Jarkko Rapala,
Docent Christina Lyra and Academy Professor Kaarina Sivonen for all the time and effort
they have invested in this work. Without Jarkko’s ideas and trust in me I probably would
never even have began this expedition and without his and Christina’s advice and support
I would still be wandering in the wilderness. Kaarina kindly took me under her wing just
when it seemed that the journey would be cut short. Her care for the wellbeing of her group
members and even strays like me has greatly helped in restoring my faith in human kind.
The three of you will always have a special place in my memories.
Professors Mirja Salkinoja-Salonen and Kristina Lindström are thanked for their help
and for instructions they have given as well as for the inspiring example they have set.
Professor Marja-Liisa Hänninen and Docent Stefan Bertilsson are sincerely thanked for
reviewing the thesis manuscript and for their most valuable comments on improving it.
I heartily thank all the co-workers at the Finnish Environment Institute for their
assistance and friendliness. Especially Kaisa Heinonen, Minna Madsen, Liisa Lepistö,
Maija Niemelä and Reija Jokipii were invaluable for the implementation of this work.
However, all of you, from the old timers like Maarit Niemi, Tuula Ollinkangas and Sinikka
Pahkala to summer trainees valuably aided this work, whether in the form of work effort
and advice in the laboratory or friendly conversations over a cup of coffee.
I would also like to acknowledge the members of the cyano group for their friendly
welcome and their generous help. A special thanks goes to Anne Ylinen, Kaisa Rantasärkkä
and Hanna Sinkko for their advice and plentiful support they have given since the day I
arrived.
All the co-authors are warmly thanked for their contributions to the articles. Jarkko,
Christina and Kaarina again deserve my special gratitude for the kind schooling they
gave me on the many aspects of writing a scientific article. I could not have had BETTER
mentors.
Partners in cooperation are acknowledged for their many contributions in collecting
and analysing the studied material. This work would have been quite skeletal without you.
I am also deeply grateful to my relatives and friends of all the encouragement they have
given over the years. You are the backbone that supports me.
My men Mikko and Leo then, there are no words strong enough to tell how much you
mean to me. As captivating as this experience has been, without you it would in many
ways have been an empty journey through a barren land.
To my valiant princes
Helsinki, November 2009
38
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51
Appendix 1
Appendix 1
The bacterial strains analysed in this study, water type of their origin, isolation medium
used, length of the acquired 16S rRNA gene sequence and its accession number, and the
taxonomic assignment of the strains.
52
Acquired 16S rRNA gene
sequence (bp)
Isolation medium
CYA
CYA
CYA
BA
BA
CYA
R2A
R2A
CYA
R2A
R2A
CYA
Z8
Z8
Z8
Z8
CYA
CYA
CYA
CYA
CYA
CYA
872
791
838
830
832
740
836
833
833
829
833
778
868
868
868
868
741
889
823
734
826
819
Taxonomic assignment
(phylum; class)
FW
FW
FW
FW
FW
FW
TW
TW
FW
TW
TW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
Gene bank accession
number
JO92
RU7
ET4
HE90
HE95
LO10
ES1
RA1
TA6A
ES10
KK5
LO12
JO69
JO70
JO72
JO75
EN4
ET11
LO2
LO4
LO5
LO6
Water type
Bacterial strain
Water type: FW refers to freshwater sample taken from Finnish lake, river or pond
water, TW to sample of treated drinking water, and BW to brackish water sample taken
from the Baltic Sea.
Isolation medium: The BA refers to blood agar, the CYA to growth medium with frozen
and thawed culture of non-microcystin-producing Anabaena strain, the R2A to Reasoner’s 2A medium, the SAA to starch ampicillin agar, the TOX to medium with demethyl
variants of microcystins and the Z8 to nutrient poor medium.
AM988866
AM988867
AM988868
AM988870
AM988871
AM988872
AM988869
AM988873
AM988874
AM988875
AM988876
AM988877
AM988878
AM988879
AM988880
AM988881
AM988882
AM988883
AM988885
AM988886
AM988887
AM988888
Actinobacteria;
Actinobacteria
Classified at genus level
Arthrobacter
Kocuria
Microbacterium
Micrococcus
Micrococcus
Micrococcus
Micrococcus
Micrococcus
Mycobacterium
Mycobacterium
Nocardia
Nocardioides
Rhodococcus
Rhodococcus
Rhodococcus
Rhodococcus
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Appendix 1
LO13
LO14
PU7
TE6
TE13
TE14
JO77
TE37
TE41
FW
FW
FW
FW
FW
FW
FW
FW
FW
CYA
CYA
CYA
CYA
CYA
CYA
R2A
TOX
TOX
701
823
821
830
831
869
869
825
833
AM988889
AM988890
AM988891
AM988892
AM988893
AM988894
AM988884
AM988895
AM988896
RU14
FW
CYA
829
AM988897
HE85
HE92
AE103
AE107
AE109
AE110
AE118
AE120
JO2
JO7
VA6
PU5
KU12
RU8
RU9
HA4
JO5
JO9
JO11
JO12
JO15
JO16
JO23
JO24
JO25
JO26
JO27
JO28
JO31
JO32
JO36
JO41
JO104
RJ1
RJ5
TE19
TE25
TE26
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
BA
BA
SAA
SAA
SAA
SAA
SAA
SAA
TOX
TOX
R2A
CYA
R2A
R2A
R2A
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
839
849
456
456
456
456
456
456
881
882
843
835
841
838
831
837
838
851
791
821
841
879
834
878
838
840
882
835
879
889
838
879
878
832
834
827
826
841
AM988898
AM988899
EU724056
EU724051
EU724052
EU724053
EU724054
EU724055
AM988900
AM988901
AM988902
AM988924
AM988923
AM988927
AM988928
AM988903
AM988904
AM988905
AM988906
AM988907
AM988908
AM988909
AM988910
AM988911
AM988912
AM988913
AM988914
AM988915
AM988916
AM988917
AM988918
AM988919
AM988920
AM988925
AM988926
AM988929
AM988930
AM988931
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Streptomyces
Classified at family level
Microbacteriaceae
Bacteroidetes;
Flavobacteria
Classified at genus level
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Chryseobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
53
Appendix 1
TE28
TE31
TE40
KK2
KK3
TE18
FW
FW
FW
TW
TW
FW
TOX
TOX
TOX
R2A
R2A
TOX
840
835
824
841
840
845
AM988932
AM988933
AM988934
AM988921
AM988922
AM988935
HE91
FW
BA
870
AM988936
OT2
TE7
JO107
TE29
RU12
JO14
JO108
LI1
JO45
PU10
TA2
TE12
IJ6
JO4
PU13
TE5
TE20
TE20B
TE23
JO56
JO115
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
CYA
CYA
TOX
TOX
CYA
TOX
TOX
CYA
CYA
CYA
CYA
CYA
TOX
TOX
TOX
TOX
TOX
TOX
TOX
Z8
TOX
792
831
869
803
842
888
884
809
840
837
837
884
781
884
794
886
833
837
839
882
862
AM988938
AM988939
AM988937
AM988940
AM988943
AM988941
AM988942
AM988944
AM988947
AM988949
AM988951
AM988953
AM988945
AM988946
AM988950
AM988952
AM988954
AM988955
AM988956
AM988948
AM988957
RU1
FW
TOX
778
AM988958
HE105
4285C
4414
HE99
ET1
RJ4
TE36
ES2
ES8
RN13
4361A
HE53
ES4
4337D
BW
FW
FW
FW
FW
FW
FW
TW
TW
TW
BW
FW
TW
FW
BA
BA
BA
BA
R2A
TOX
TOX
R2A
R2A
R2A
BA
BA
R2A
BA
496
496
496
496
858
816
903
863
859
849
495
861
833
497
AM988965
AM988959
AM988960
AM988964
AM988963
AM988966
AM988968
AM988961
AM988962
AM988967
AM988969
AM988970
AM988971
AM988972
54
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Flavobacterium
Riemerella
Classified at family level
Flavobacteriaceae
Bacteroidetes;
Sphingobacteria
Classified at genus level
Arcicella
Arcicella
Arcicella
Arcicella
Chitinophaga
Chitinophaga
Chitinophaga
Flectobacillus
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Pedobacter
Spirosoma
Deinococcus-Thermus;
Deinococci
Classified at genus level
Deinococcus
Firmicutes; Bacilli
Classified at genus level
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Bacillus
Caryophanon
Lactococcus
Paenibacillus
Staphylococcus
Appendix 1
4346A
JO48
SL5
FW
FW
TW
BA
CYA
R2A
497
903
844
AM988973
AM988975
AM988974
KU2
TE21
JO50
RN8
RN9
HA3
LO7
RU13
TE8
TE10
TE11
TE34
KK9
RA3
RN17
4363A
JO43
JO46
PU6
RU6
KU11
JO6
JO103
JO62
HV3
KK4
RA10
SL1
SL2A
SL2B
VA7
ET2
JO49
JO91
JO95
PU8
TE1
JO76
JO100
JO51
EN3
EN5
ET7
ET10
LO11
OT1
FW
FW
FW
TW
TW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
TW
TW
TW
TW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
R2A
TOX
Z8
R2A
R2A
CYA
CYA
CYA
CYA
CYA
CYA
TOX
R2A
R2A
R2A
BA
CYA
CYA
CYA
CYA
R2A
TOX
TOX
Z8
R2A
R2A
R2A
R2A
R2A
R2A
R2A
CYA
CYA
CYA
CYA
CYA
CYA
R2A
TOX
Z8
CYA
CYA
CYA
CYA
CYA
CYA
805
781
836
840
744
779
790
774
797
795
800
795
791
799
794
442
837
839
790
808
779
836
837
840
838
780
771
778
779
779
836
793
838
839
837
799
803
840
840
838
787
790
750
849
788
792
AM988977
AM988978
AM988976
AM988979
AM988980
AM988981
AM988983
AM988986
AM988987
AM988988
AM988989
AM988990
AM988982
AM988984
AM988985
AM988991
AM988994
AM988995
AM989000
AM989002
AM988999
AM988993
AM988997
AM988996
AM988992
AM988998
AM989001
AM989003
AM989004
AM989005
AM989006
AM989007
AM989008
AM989011
AM989012
AM989014
AM989015
AM989010
AM989013
AM989009
AM989016
AM989017
AM989022
AM989023
AM989025
AM989026
Staphylococcus
Staphylococcus
Staphylococcus
Proteobacteria;
Alphaproteobacteria
Classified at genus level
Azospirillum
Azospirillum
Azospirillum
Blastomonas
Blastomonas
Bosea
Bosea
Bosea
Bosea
Bosea
Bosea
Bosea
Bosea
Bosea
Bosea
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Brevundimonas
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Caulobacter
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
55
Appendix 1
OT4
OT5
OT7
PU2
PU3
TA3
TA5
KU9
ES3
ES5
ES6
ES11
ET5
TA4A
TA1
LO3
HV6
HV9
VA5
KU5
IJ2
IJ5
ET6
JO47
JO78
JO79
JO89
LI2
LO8
TA2A
TU6A
TU3
JO3
JO8
HV2
HV4
HV7
HV10
RA5
RA7
RN2
SL7
SL9
VA4
VA8
KU13
JO106
RA4
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
TW
FW
FW
FW
FW
TW
TW
TW
BW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
TW
TW
TW
TW
TW
TW
TW
TW
FW
FW
TW
CYA
CYA
CYA
CYA
CYA
CYA
CYA
R2A
R2A
R2A
R2A
R2A
CYA
CYA
R2A
CYA
R2A
R2A
R2A
R2A
CYA
TOX
CYA
CYA
CYA
CYA
CYA
CYA
CYA
CYA
CYA
R2A
TOX
TOX
R2A
R2A
R2A
R2A
R2A
R2A
R2A
R2A
R2A
R2A
R2A
R2A
TOX
R2A
771
742
750
757
757
737
736
875
794
799
800
799
795
736
788
809
837
776
835
748
792
802
750
840
839
792
794
840
805
788
798
788
840
817
841
786
832
839
788
789
788
788
803
796
841
787
841
789
AM989027
AM989028
AM989029
AM989030
AM989031
AM989032
AM989033
AM989024
AM989018
AM989019
AM989020
AM989021
AM989034
AM989036
AM989035
AM989037
AM989038
AM989039
AM989040
AM989041
AM989042
AM989043
AM989044
AM989051
AM989052
AM989053
AM989054
AM989055
AM989056
AM989062
AM989064
AM989063
AM989049
AM989050
AM989045
AM989046
AM989047
AM989048
AM989057
AM989058
AM989059
AM989060
AM989061
AM989065
AM989066
AM989068
AM989067
AM989069
TA7
TA7A
RA2
RN19
FW
FW
TW
TW
CYA
CYA
R2A
R2A
803
788
788
842
AM989070
AM989073
AM989071
AM989072
56
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Methylobacterium
Novosphingobium
Novosphingobium
Novosphingobium
Paracoccus
Phyllobacterium
Phyllobacterium
Phyllobacterium
Porphyrobacter
Sphingobium
Sphingobium
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingomonas
Sphingopyxis
Sphingopyxis
Sphingopyxis
Classified at family level
Acetobacteraceae
Sphingomonadaceae
Sphingomonadaceae
Sphingomonadaceae
Appendix 1
KU10
TA3A
FW
FW
15Ba
2a
2B15a
2B17a
2B3a
S1030 a,b
G44a
F39a
F36 a,b
7Aa
2C23a,b
2C20T a,b
2B5a
KU6
HE147
4398A
TA13
4288A
4336A
HE106
HE127
HE160
HA1
LO1
TA4
TA11
JO22
JO73
KK1
KK11
EN2
JO80
JO83
LO9
PU4
HA5
JO114
TE24
JO59
KK10
RN14
RN18
PU12
JO1
BW
FW
BW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
FW
FW
R2A
CYA
793
800
AM989074
AM989075
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
TOX
1422
1423
1431
1428
1421
1428
1434
1425
1430
1414
1444
1429
1384
AY515379
AY515380
AY515381
AY515382
AY515383
AY515384
AY515385
AY515386
AY515387
AY515388
AY515389
AY515390
AY515391
R2A
BA
BA
TOX
BA
BA
BA
BA
BA
CYA
CYA
CYA
CYA
TOX
TOX
R2A
R2A
CYA
CYA
CYA
CYA
CYA
TOX
TOX
TOX
Z8
R2A
R2A
R2A
TOX
TOX
837
483
413
886
483
483
483
474
485
850
839
903
848
807
871
823
823
851
891
893
846
843
848
888
848
855
839
693
695
802
890
AM989076
AM989077
AM989078
AM989079
AM989080
AM989081
AM989082
AM989083
AM989084
AM989085
AM989090
AM989091
AM989092
AM989086
AM989087
AM989088
AM989089
AM989093
AM989096
AM989097
AM989100
AM989101
AM989094
AM989098
AM989102
AM989095
AM989099
AM989103
AM989104
Classified at class level
Alphaproteobacteria
Alphaproteobacteria
Proteobacteria;
Betaproteobacteria
Classified at species
level
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Paucibacter toxinivorans
Classified at genus level
Aquabacterium
Aquitalea
Burkholderia
Chitinimonas
Chromobacterium
Chromobacterium
Chromobacterium
Chromobacterium
Chromobacterium
Duganella
Duganella
Duganella
Duganella
Duganella
Duganella
Duganella
Duganella
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Herbaspirillum
Iodobacter
Janthinobacterium
57
Appendix 1
ET8
ET9
HA2
JO84
JO86
JO99
TE4
IJ3
JO87
JO90
JO96
JO97
KU4
KU7
TE15
TE17
TE22
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
CYA
CYA
CYA
CYA
CYA
CYA
TOX
CYA
CYA
CYA
CYA
CYA
R2A
R2A
TOX
TOX
TOX
847
886
833
830
835
880
843
839
832
881
829
854
835
817
832
849
851
AM989105
AM989106
AM989107
AM989108
AM989109
AM989110
AM989111
AM989112
AM989113
AM989114
AM989115
AM989116
AM989117
AM989118
AM989119
AM989120
AM989121
KU8
JO111
KU1
JO21
JO113
JO53
KK7
RN12
FW
FW
FW
FW
FW
FW
TW
TW
R2A
TOX
R2A
TOX
TOX
Z8
R2A
R2A
838
880
851
797
891
889
847
806
AM989122
AM989123
AM989128
AM989124
AM989126
AM989125
AM989127
AM989129
KU14
FW
R2A
805
AM989130
4286C
4300B
4362A
4367I
HE21
HE55
HE120
HE124
HE128
HE138
HE139
HE140
HE144
HE145
HE146
AU2
AU3
JO101
RU5
TE16
SL10
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
R2A
R2A
TOX
TOX
TOX
R2A
485
485
483
485
856
853
485
485
485
488
483
483
487
487
487
849
847
895
853
850
851
AM989131
AM989132
AM989133
AM989134
AM989137
AM989138
AM989139
AM989140
AM989141
AM989142
AM989143
AM989144
AM989145
AM989146
AM989147
AM989135
AM989136
AM989148
AM989149
AM989151
AM989150
58
Pelomonas
Pelomonas
Pelomonas
Pelomonas
Pelomonas
Pelomonas
Rhodoferax
Roseateles
Roseateles
Roseateles
Roseateles
Roseateles
Roseateles
Roseateles
Vogesella
Vogesella
Vogesella
Classified at family level
Alcaligenaceae
Incertae sedis 5
Oxalobacteraceae
Oxalobacteraceae
Oxalobacteraceae
Oxalobacteraceae
Oxalobacteraceae
Oxalobacteraceae
Classified at class level
Betaproteobacteria
Proteobacteria;
Gammaproteobacteria
Classified at genus level
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Acinetobacter
Appendix 1
4218B
4218E
4282D
4282J
4294H
4295A
4295F
4298D
4329A
4330A
4330D
4330E
4330G
4348A
4348D
4359B
4368D
4387A
4387B
HE1
HE6
HE7
HE34
HE35
HE94
HE112
HE125
HE130
HE132
HE136
HE149
HE154
HE163
AE26
AE27
AE28
AE30
AE31
AE32
AE33
AE34
AE35
AE36
AE37
AE39
AE40
AE41
AE42
AE43
AE44
AE45
AE59
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
492
492
492
492
492
492
491
491
492
489
492
492
494
491
494
492
492
492
492
867
861
864
857
857
856
492
492
489
489
492
492
492
492
468
452
468
453
468
444
468
468
468
468
468
468
468
468
454
468
468
468
468
AM989159
AM989160
AM989161
AM989162
AM989166
AM989167
AM989168
AM989169
AM989182
AM989183
AM989184
AM989185
AM989186
AM989188
AM989189
AM989190
AM989193
AM989194
AM989195
AM989198
AM989201
AM989202
AM989218
AM989219
AM989250
AM989253
AM989257
AM989258
AM989259
AM989260
AM989261
AM989263
AM989268
EU724006
EU724007
EU723962
EU723973
EU723952
EU723963
EU723902
EU723977
EU723956
EU724009
EU724010
EU723964
EU723967
EU723958
EU724011
EU724012
EU723968
EU723972
EU723978
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
59
Appendix 1
AE60
AE61
AE62
AE63
AE64
AE65
AE67
AE72
AE73
AE74
AE81
AE83
AE84
AE85
AE86
AE87
AE88
AE89
AE90
AE91
AE95
AE131
AE137
AE138
AE139
AE141
AE158
AE169
AE170
AE171
AE172
AE174
AE175
AE176
AE188
AE189
AE191
AE192
AE246
AE248
AE249
AE250
AE251
4192A
4201B
4201D
4201G
4207A
4207C
4207D
4286A
4286B
60
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
FW
FW
FW
FW
FW
FW
FW
FW
FW
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
BA
BA
BA
BA
BA
BA
BA
BA
BA
468
468
468
468
468
468
468
468
468
431
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
468
452
468
468
468
468
468
468
431
468
468
468
468
491
492
492
492
492
492
492
492
492
EU723979
EU724013
EU724014
EU724015
EU723989
EU723994
EU724016
EU724017
EU724018
EU723912
EU724021
EU723991
EU724022
EU723993
EU724023
EU724024
EU724025
EU724026
EU724027
EU723999
EU724028
EU723948
EU723981
EU723982
EU724045
EU724031
EU724046
EU723965
EU723983
EU723960
EU723974
EU723908
EU723893
EU723900
EU723995
EU723961
EU724035
EU723992
EU723913
EU723996
EU724044
EU723987
EU723997
AM989152
AM989153
AM989154
AM989155
AM989156
AM989157
AM989158
AM989163
AM989164
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Appendix 1
4287D
4300D
4300E
4301B
4301C
4301D
4302G
4316E
4316F
4318A
4318B
4318C
4326G
4337B
4365A
4366A
4400A
4402C
HE3
HE4
HE9
HE10
HE11
HE12
HE13
HE14
HE15
HE16
HE17
HE18
HE19
HE20
HE22
HE28
HE29
HE37
HE38
HE39
HE40
HE44
HE45
HE46
HE47
HE48
HE51
HE56
HE57
HE60
HE61
HE62
HE63
HE64
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
492
491
492
492
492
492
492
492
492
492
905
492
492
492
492
492
492
492
856
855
864
861
859
863
842
863
857
864
865
855
856
863
864
860
856
856
856
858
851
855
851
855
857
864
859
855
858
855
858
856
855
863
AM989165
AM989170
AM989171
AM989172
AM989173
AM989174
AM989175
AM989176
AM989177
AM989178
AM989179
AM989180
AM989181
AM989187
AM989191
AM989192
AM989196
AM989197
AM989199
AM989200
AM989203
AM989204
AM989205
AM989206
AM989207
AM989208
AM989209
AM989210
AM989211
AM989212
AM989213
AM989214
AM989215
AM989216
AM989217
AM989220
AM989221
AM989222
AM989223
AM989224
AM989225
AM989226
AM989227
AM989228
AM989229
AM989230
AM989231
AM989232
AM989233
AM989234
AM989235
AM989236
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
61
Appendix 1
HE65
HE73
HE74
HE75
HE76
HE77
HE78
HE79
HE80
HE82
HE83
HE86
HE89
HE101
HE110
HE113
HE114
HE115
HE150
HE155
HE157
HE158
HE159
HE165
TU4
TU6
AE1
AE2
AE3
AE6
AE7
AE9
AE10
AE12
AE13
AE14
AE15
AE16
AE17
AE18
AE19
AE20
AE21
AE23
AE25
AE48
AE53
AE71
AE75
AE78
AE92
AE93
62
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
CYA
CYA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
864
859
866
856
859
858
856
857
863
864
858
858
858
491
492
492
491
492
492
492
492
492
492
492
850
807
481
468
443
468
468
468
468
468
468
468
451
468
443
468
452
468
468
468
468
448
468
468
468
468
468
445
AM989237
AM989238
AM989239
AM989240
AM989241
AM989242
AM989243
AM989244
AM989245
AM989246
AM989247
AM989248
AM989249
AM989251
AM989252
AM989254
AM989255
AM989256
AM989262
AM989264
AM989265
AM989266
AM989267
AM989269
AM989271
AM989272
EU724000
EU723949
EU723887
EU724001
EU724048
EU723888
EU724002
EU724003
EU724004
EU723889
EU724008
EU723985
EU723907
EU723953
EU724005
EU723905
EU723998
EU723918
EU723906
EU723947
EU723927
EU723954
EU724019
EU724020
EU723970
EU723915
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Appendix 1
AE98
AE99
AE101
AE102
AE104
AE105
AE106
AE111
AE117
AE119
AE121
AE122
AE123
AE125
AE126
AE127
AE128
AE132
AE133
AE143
AE145
AE147
AE150
AE152
AE153
AE154
AE161
AE163
AE165
AE167
AE168
AE180
AE181
AE194
AE195
AE196
AE197
AE198
AE199
AE200
AE201
AE202
AE203
AE204
AE205
AE206
AE207
AE208
AE209
AE210
AE213
AE214
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
468
468
468
468
468
468
468
468
452
433
468
468
443
443
443
468
443
443
443
468
468
468
450
468
468
468
468
468
468
467
468
468
468
468
468
468
468
446
468
468
443
468
468
468
468
468
468
468
468
468
468
468
EU723932
EU723933
EU723934
EU723935
EU723896
EU723938
EU724029
EU724030
EU723984
EU723916
EU723890
EU723919
EU723895
EU723897
EU723910
EU723898
EU723899
EU723891
EU723892
EU723988
EU723955
EU723959
EU723931
EU723942
EU724032
EU723929
EU723950
EU723951
EU723980
EU724033
EU724034
EU723925
EU723976
EU724036
EU723901
EU723920
EU724037
EU723917
EU724038
EU724039
EU723909
EU723936
EU723969
EU723921
EU723975
EU723894
EU723923
EU724040
EU724041
EU723926
EU724042
EU723914
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
63
Appendix 1
AE215
AE216
AE217
AE218
AE219
AE222
AE223
AE230
AE232
AE233
AE234
AE235
AE237
AE238
AE239
AE240
AE243
AE256
AE258
AE259
AE260
JO110
AE162
4329C
HE5
HE49
HE54
HE108
HE116
HE2
HE8
HE23
HE24
HE27
HE66
HE67
HE68
HE69
HE70
HE71
HE93
HE103
RU10
KU3
JO33
TE27
RN1
RN5
RN21
SL3
SL4
SL8
64
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
BW
BW
BW
BW
BW
BW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
FW
TW
TW
TW
TW
TW
TW
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
SAA
TOX
SAA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
CYA
R2A
TOX
TOX
R2A
R2A
R2A
R2A
R2A
R2A
468
468
445
433
468
468
468
468
468
468
468
445
468
468
468
468
468
443
468
443
468
896
441
482
856
851
847
484
482
858
856
857
857
848
850
851
848
849
848
849
847
484
813
852
888
848
888
888
889
850
834
852
EU723928
EU723924
EU723903
EU723930
EU723937
EU723990
EU723940
EU724047
EU724049
EU724043
EU723939
EU723943
EU723922
EU723946
EU723986
EU723945
EU723966
EU723904
EU723971
EU723911
EU723957
AM989270
EU724057
AM989273
AM989275
AM989280
AM989281
AM989290
AM989291
AM989274
AM989276
AM989277
AM989278
AM989279
AM989282
AM989283
AM989284
AM989285
AM989286
AM989287
AM989288
AM989289
AM989297
AM989293
AM989292
AM989302
AM989294
AM989295
AM989296
AM989298
AM989299
AM989300
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Aeromonas
Erwinia
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Pseudomonas
Appendix 1
SL11
VA2
4218A
4218G
TE32
HE59
TE38
HE30
HE32
HE33
HE36
HE42
HE43
HE50
HE87
HE88
HE111
HE161
HE162
TW
TW
BW
BW
FW
FW
FW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
BW
R2A
R2A
BA
BA
TOX
BA
TOX
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
BA
850
889
482
483
838
857
855
855
864
864
855
854
852
854
853
853
487
490
489
AM989301
AM989303
AM989304
AM989305
AM989306
AM989307
AM989308
AM989309
AM989310
AM989311
AM989312
AM989313
AM989314
AM989315
AM989316
AM989317
AM989318
AM989319
AM989320
Pseudomonas
Pseudomonas
Rheinheimera
Rheinheimera
Rheinheimera
Serratia
Stenotrophomonas
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Vibrio
Classified at family level
HE25
FW
BA
854
AM989321
Enterobacteriaceae
HE26
FW
BA
855
AM989322
Enterobacteriaceae
HE58
FW
BA
860
AM989323
Enterobacteriaceae
HE72
FW
BA
852
AM989324
Enterobacteriaceae
AE51
FW
SAA
397
EU724058
Enterobacteriaceae
AE164
FW
SAA
452
EU724059
Enterobacteriaceae
4218F
BW
BA
492
AM989325
Vibrionaceae
a
Paucibacter toxinivorans strains were originally isolated by Lahti et al. (1998) and were shown
to degrade microcystins in their preliminary studies, using mainly one demethyl variant of microcystin-RR. The strains G44 and F36 were shown to degrade also nodularin in preliminary
studies.
b
The strain was found to degrade microcystins LR and YR as well as nodularin in this study.
65