Heterotrophic Bacteria Associated with Cyanobacteria in Recreational and Drinking Water

Katri Berg

Department of Applied Chemistry and MicrobiologyDivision of Microbiology

Faculty of Agriculture and ForestryUniversity 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 RapalaNational Supervisory Authority for Welfare and Health, Finland Docent Christina LyraDepartment of Applied Chemistry and MicrobiologyUniversity of Helsinki, Finland Academy Professor Kaarina SivonenDepartment of Applied Chemistry and MicrobiologyUniversity of Helsinki, Finland

Reviewers:Professor Marja-Liisa HänninenDepartment of Food and Environmental HygieneUniversity of Helsinki, Finland

Docent Stefan Bertilsson Department of Ecology and Evolution Uppsala University, Sweden

Opponent:Professor Agneta AnderssonDepartment of Ecology and Environmental SciencesUmeå University, Sweden

Printed Yliopistopaino, Helsinki, 2009Layout Timo Päivärinta

ISSN 1795-7079ISBN 978-952-10-5892-9 (paperback)ISBN 978-952-10-5893-6 (PDF)

e-mail katri.berg@helsinki.fi

Front cover pictureDAPI 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 identifi cation 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 effi ciency ................................................................... 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 effi ciency .................................................................... 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 identifi cation. 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 fl agellae 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 identifi cation of the strains.

ABBREVIATIONS

Adda (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10- phenyldeca-4,6-dienoic acidAH Aeromonas hydrophilaBA Blood agarBLAST Basic local alignment search toolCAP Program for canonical analysis of principal coordinates CFU Colony forming unitDISTLMf Program for distance-based multivariate analysis for a linear model using forward selectionELISA Enzyme-linked immunosorbent assay EU Endotoxin unitGLC Gas-liquid chromatography HPLC High-performance liquid chromatography LD50 Lethal dose, 50%Microcystin-LR Microcystin with leusine as aminoacid 2 and arginine as amino acid 4Microcystin-YR Microcystin with tyrosine as aminoacid 2 and arginine as amino acid 4NCBI National Center for Biotechnology InformationPAUP Phylogenetic analysis using parsimonyPCO Program for principal coordinate analysisPCR Polymerase chain reactionPHYLIP Phylogeny inference packageR2A Reasoner’s 2A mediumSAA Starch ampicillin agarTSA Trypticase soy agar UPGMA Unweighted pair group method with arithmetic averagesWHO 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 purifi cation effi ciency 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), fl agellin subunits (fl aA/fl aB), 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 fl a, 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 purifi cation effi ciency 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 effi ciently 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 biofi lm 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 iho- sekä 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 heterotrofi set 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 heterotrofi sta 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 heterotrofi sten bakteerien yhteisö.

Eristetyistä heterotrofi sista 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), fl agelliini-alayksikköjä (fl aA/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 fl a, 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 heterotrofi set 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 heterotrofi sten bakteerien määrät vähenivät vedenkäsittelyn aikana tehokkaasti. Sen sijaan pienet planktonsolut läpäisivät vedenkäsittelyketjun säännöllisesti. Puhdistusketjun läpäisevät solut voivat edistää biofi lmien muodostumista vedenjakeluverkostossa ja siten heikentää käsitellyn veden laatua.

Työssä käsitellyt heterotrofi set 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 heterotrofi set bakteerit pitäisi ottaa huomioon syanobakteerikukintoihin liittyvien terveysriskien arvioinnissa.

1

1. INTRODUCTION

1.1 CyanobacteriaCyanobacteria 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 fi lamentous 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 fi laments 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 fi eld 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, fl oating 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 cyanobacteriaThe water blooms can be produced by various cyanobacterial species within

Introduction

2

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 fl uxes for photosynthesis.

1.3 Cyanobacterial toxinsCyanobacteria produce a wide variety of secondary metabolites, many of them with so far unknown functions. The metabolites

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 HepatotoxinsThe 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-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-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

3

(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 specifi c uptake system largely restricts the effect of the toxins in mammals to the liver, which gives them the classifi cation 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 NeurotoxinsBoth 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 (Kuiper-Goodman 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). Anatoxin-a(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 shellfi sh poisons (Sivonen and Börner 2008). They include nonsulphated (saxitoxin and neosaxitoxin), singly sulphated (gonyautoxins) and doubly sulphated (C-toxins) compounds.

Introduction

4

Introduction

Of these compounds, saxitoxin is the most toxic. Saxitoxins were originally identifi ed in marine dinofl agellates. 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 shellfi sh 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 fi ndings 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,

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 cyanobacteriaExamples 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 Cyl indrospermopsis , 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 (Kuiper-Goodman 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.

5

Introduction

The most signifi cant route of exposure in association with cyanobacterial blooms is probably by inhalation (Stewart et al. 2006a, Anderson et al. 2007).

1.3.4 Toxin analysesThe 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 specifi city, 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 high-performance 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 anatoxin-a(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, specifi city 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.

6

Introduction

1.3.5 Persistence of cyanobacterial toxinsThe 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

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 effl uent 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 Adda-Arginine 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

7

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 fi xed by the primary producers and form an important part of the aquatic nutrient web.

1.4.1 Aquatic bacteriaBased 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 Alpha- and 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 Gamma- and 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 fi x 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

8

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 vulnifi cus abundances. Specifi c 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 benefi t 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 benefi t 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

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 benefi t 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 characterisationInterpretation 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

9

Introduction

those supported by the chosen cultivation conditions.

1.5.1 Isolation of bacteriaThe 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 artifi cial 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 identifi cation 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 identifi cation of bacteria of specifi c metabolic types. For example, the API 20 NE test kit for the identifi cation of non-fastidious, non-enteric, Gram-negative 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 ZymProfi ler® fl uorogenic analysis designed by Vepsäläinen et al. (2001) can be used for the screening of enzymatic activities of bacteria from purifi ed or complex samples (bioMèrieux 2009, Tharagonnet et al. 1977, Allen et

10

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 identifi cation 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 identifi cation 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 classifi er and sequence match tools provided by the Ribosomal Database Project II.

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 neighbour-joining, 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 confi dence test such as bootstrapping, and by comparing the trees obtained by using different tree building methods (Nei and Kumar 2000a, Felsenstein 2004a).

Introduction

11

Introduction

1.5.4 Genetic analyses in assessment of physiological featuresSince the physiological features of the bacteria are ultimately based on their gene content, molecular methods such as PCR with gene specifi c 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 fl agella (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 infl ated 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

12

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 bloomsEutrofication 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 fi sh, 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, insuffi cient 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).

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

13

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, Martin-Carnahan 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. popoffi i 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. vulnifi cus 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.

14

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 suffi cient water quality despite the normal performance fl uctuations 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 specifi c 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 purifi cation effi ciency of different treatment combinations in a real

Introduction

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 treatmentIf surface water is used, the water treatment steps usually include coagulation, fl occulation and clarifi cation (by sedimentation or fl otation) followed by fi ltration 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 effi ciency and the effi cient 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 effi ciently 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 effi ciency 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, clarifi cation and fi ltration 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 effi ciently remove cyanobacterial toxins

15

Introduction

within those cells (Hitzfeld et al. 2000, Drikas et al. 2001, Svrcek and Smith 2004). In contrast, coagulation and clarifi cation are quite ineffi cient against toxins already released from the cells into water. Filtration, especially with slow sand fi lters 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 effi ciently 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 fi ltration 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 effi cient 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 purifi cation steps. For example, in the fi ltration step rapid gravity and pressure fi lters or slow sand fi lters can be used (Svrcek and Smith 2004, LeChevallier and Au 2004d, WHO 2008b). Additional steps such as pre-treatment of the water with bank fi ltration 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). Effi cient 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 qualityIn 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

16

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 suffi ciently 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 CO2 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 biofi lm 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). Biofi lms 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

Introduction

Legionella, Mycobacterium, Aeromonas or Pseudomonas (Percival and Walker 1999, Theron and Cloete 2002, Berry et al. 2006, WHO 2008c and e). The biofi lms 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 biofi lms 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 fi lters. Furthermore, a Sphingopyxis strain that degraded microcystins was isolated from such a fi lter (Ho et al. 2007). The studies showed that the heterotrophic bacteria adapted to

17

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 fi lters. 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 effi cient use of such applications.

18

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 effi ciency 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 purifi cation effi ciency of a modern drinking water treatment process at an operating water treatment plant, during mass occurrence of cyanobacteria in the source water (IV).

19

Materials and Methods

3. MATERIALS AND METHODS

3.1 Water samples and isolated bacterial strainsWater 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 identifi ed in the original articles (I, II, IV). In addition, 13 previously isolated hepatotoxin degrading Betaproteobacteria strains were studied (III).

3.2 MethodsThe 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 studyMethod PaperSampling and strain isolation I, II, IVa

Telephone interviews for collecting data on adverse human health effects IIPhytoplankton identifi cation and biomass analysis IVGram staining I, II, III, IV Flagella staining IIIAPI NE and API ZYM tests IIIZymProfi ler® test IIIGN MicroPlate test IIIFatty acid analysis by gas-liquid chromatography (GLC) IIIBacterioclorophyll a analysis by spectrophotometry IIIOxidase and catalase tests II, IIIAeromonas hydrophila (AH) medium tests IIMotility on casitone-yeast extract agar IIIHaemolytic activity on blood agar ITesting of the effects of the isolates on cyanobacterial growth IHepatotoxins by enzyme-linked immunosorbent assay (ELISA) II, IVHepatotoxins by protein phosphatase inhibition assay III, IVEndotoxins by kinetic chromogenic Limulus amoebocyte lysate assay II, IVOverall toxicity by Artemia salina bioassay IVHepatotoxin degradation test IIIAnatoxin-a by high-performance liquid chromatography (HPLC) II, IVb

Saxitoxins by Jellett Rapid paralytic shellfi sh poisons test strips II, IVc

Putative presence of anatoxin-a(S) by acetylcholine esterase inhibition test II, IVd

DNA extraction from bacterial strains I, II, III, IVe

16S rRNA gene amplifi cation and sequencing I, II, III, IVf

Genomic guanine-cytosine content by high-performance liquid chromatography (HPLC)

III

Taxonomic classifi cation by basic local alignment search tool (BLAST) I, III, IV

20

Method PaperTaxonomic classifi cation by taxonomic classifi er and sequence match tools I, II16S rRNA gene sequence similarity analysis by BioEdit II, IIIPhylogenetic analysis with neighbour-joining and maximum parsimony criteria IIIVisualization of a priori grouping by principal coordinate analysis IIScreening of Aeromonas virulence genes by PCR IIMultivariate multiple regression analysis IICanonical discriminant analysis II

aStrain isolation was done as described in I.bThe presence of anatoxin-a, homoanatoxin-a, and their epoxy and dihydro degradation products were analysed as described by James et al. (1997).cThe presence of saxitoxins was analysed using the commercial Jellett Rapid paralytic shellfi sh poisons test strips (Jellett et al. 2002).dThe putative presence of anatoxin-a(S) was analysed as described by Ellman et al. (1961).eThe DNA extraction was executed as described in III.fSequencing was carried out at the Institute of Biotechnology of the University of Helsinki.

Table 1 cont.

Materials and Methods

21

Results

4. RESULTS

4.1 Heterotrophic bacteria associated with cyanobacteriaIn 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 fi ve 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 classifi er (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 cyano-bacterial hepatotoxins: Paucibacter toxinivorans gen. nov. sp. nov.The previously isolated 13 Betaproteo-bacteria 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.

22

Results

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.

Cla

ss

ActinobacteriaFlavobacteria

SphingobacteriaDeinococci

BacilliAlphaproteobacteriaBetaproteobacteria

Gammaproteobacteria

A Number of the strains

0 40 80 120 160 200 240 280 320 3605%

7%3%

0 2%3%

16%9%

57%

TOX CYA R2A Z8 BA SAA

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

leucine arylamidase (III: Supplementary table S1). In addition, all were positive for acid phosphatase, naphthol-AS-BI-phosphohydrolase, 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 Gram-negative, rod-shaped (0.5–0.7×1.3–5.0

23

Results

0 10 20 30 33 282121

1Actinobacteria

2Flavobacteria

3Sphingobacteria

4Deinococci

5Bacilli

6Alphaproteobacteria

7Betaproteobacteria

8Gam

maproteobacteria

Number of the strains

ArthrobacterKocuria

MicrobacteriumMicrococcus

MycobacteriumNocardia

NocardioidesRhodococcusStreptomyces

Assigned at family level

ChryseobacteriumFlavobacterium

RiemerellaAssigned at family level

ArcicellaChitinophagaFlectobacillusPedobacterSpirosoma

Deinococcus

BacillusCaryophanonLactococcusPaenibacillus

Staphylococcus

AzospirillumBlastomonas

BoseaBrevundimonas

CaulobacterMethylobacteriumNovosphingobium

ParacoccusPhyllobacteriumPorphyrobacterSphingobium

SphingomonasSphingopyxis

Assigned at family levelAssigned at class level

AquabacteriumAquitalea

BurkholderiaChitinimonas

ChromobacteriumDuganella

HerbaspirillumIodobacter

JanthinobacteriumPelomonasRhodoferaxRoseatelesVogesella

Assigned at family levelAssigned at class level

AcinetobacterAeromonas

ErwiniaPseudomonasRheinheimera

SerratiaStenotrophomonas

VibrioAssigned at family level

1

2

3

4

5

6

7

8

Clas

s(1

–8)

and

genu

sB

TOX CYA R2A Z8 BA SAATOX CYA R2A Z8 BA SAA

24

μm) and motile by a single polar fl agellum (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 cyanobacteriaHaemolytic 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

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.

Aeromonas; 92; 69%

Acinetobacter; 10; 8%

Chromobacterium; 4; 3% Burkholderia; 1; 1%Aquitalea; 1; 1%

Staphylococcus; 1; 1%Brevundimonas; 1; 1%

Bacillus; 2; 2%

Flavobacteriaceae; 1; 1%

Vibrionaceae; 1; 1%Vibrio; 11; 8%

Rheinheimera; 2; 2%Pseudomonas; 6; 5%

Bacteroidetes: Flavobacteria Firmicutes: Bacilli

Proteobacteria: Alphaproteobacteria Betaproteobacteria Gammaproteobacteria

Results

25

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 fl agellin subunits encoding fl a (fl aA/fl aB), 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, identifi ed 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 cyanobacteriaThe majori ty (60%) of the 183 heterotrophic bacterial strains analysed either enhanced or inhibited the growth of the microcystins-producing Microcystis PCC 7941 or the non-microcystin-

Results

26

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.

4.2 Selectivity of the used growth mediaThe 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-microcystin-producing 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

Figure 3: Effect of the tested heterotrophic bacterial strains on the growth of the microcystins-producing Microcystis PCC 7941 and non-microcystin-producing Anabaena PCC 7122 strains.A: at the class level. The percentages of strains with specifi c 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 identifi ed 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.

35%

29%59%

40%

25%

36%55%

60%

100%35%

54%22%

50%

58%45%

4%

25%

17%19%

25%

3%2%

3%

0 5 10 15 20 25 30 35 40 45 50 55

Cla

ss

Number of the strains

Actinobacteria

Flavobacteria

Sphingobacteria

Deinococci

Bacilli

Alphaproteobacteria

Betaproteobacteria

Gammaproteobacteria

No effect Enhanced Inhibited Enhanced/inhibited

A

27

Results

0 5 10 15 20 25 30

Clas

s (1

–8)

and

genu

s or

fam

ilyNumber of the strains

ArthrobacterKocuria

MicrobacteriumMicrococcus

NocardioidesRhodococcusStreptomyces

Microbacteriaceae

ChryseobacteriumFlavobacterium

Riemerella

ArcicellaChitinophagaFlectobacillus

PedobacterSpirosoma

Deinococcus

BacillusStaphylococcus

AzospirillumBosea

BrevundimonasCaulobacter

MethylobacteriumNovosphingobium

ParacoccusSphingobium

SphingomonasSphingopyxis

Acetobacteraceae

DuganellaHerbaspirillum

IodobacterJanthinobacterium

PelomonasRhodoferaxRoseatelesVogesella

Incertae sedis 5Oxalobacteraceae

AcinetobacterAeromonas

PseudomonasRheinheimera

Stenotrophomonas

2

1

3

4

5

6

7

8

1A

ctinobacteria 2Flavobacteria 3

Sphingobacteria 4D

einococci 5Bacilli

6A

lphaproteobacteria 7Betaproteobacteria 8

Gam

maproteobacteria

No effect Enhanced Inhibited Enhanced/inhibited

B

28

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 s trains 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 effi ciencyThe efficiency of the drinking water treatment process during cyanobacterial mass occurrence in the source water was assessed at an operating water treatment

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 fi ltration, ozonation, slow sand fi ltration 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, clarifi cation, sand fi ltration 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 fi rst 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 fi ltration reduced the endotoxins by 0.72–2.03 log10 units, and ozonation by 0.04–0.19 log10 units. The effi ciency of the

29

slow sand fi ltration 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 fi ltration steps, removed endotoxins effi ciently.

Signs of neurotoxic agent were detected by the Artemia sal ina 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), anatoxin-a(S) by acetylcholinesterase inhibition assay, and saxitoxins by Jellett rapid paralytic shellfi sh 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 fi laments (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 μm3, including possible bacterial cells) and monad cells (size class 6–65 μm3) 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 fi laments 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 fi ltration 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 effi cient removal of the cultivable heterotrophic bacteria during the water treatment process.

Results

30

Results

4.3.3 Taxonomy of the heterotrophic bacteria isolated from drinking waterThe 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 fi ve 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.

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.

Pseudomonas: 3/RN, 4/SL, 1/VA

Acinetobacter: 1/SL

Micrococcus: 1/ES, 1/RA Mycobacterium: 1/ES

Nocardia: 1/KK

Oxalobacteraceae: 1/KK, 1/RN

Chryseobacterium: 1/VA Flavobacterium: 2/KK

Bacillus: 2/ES, 1/RN Paenibacillus: 1/ES Staphylococcus: 1/SL

Blastomonas: 2/RN

Bosea: 1/KK, 1/RA, 1/RN

Brevundimonas: 1/HV, 1/KK, 1/RA,3/SL, 1/VA

Methylobacterium: 4/ES

Phyllobacterium: 2/HV, 1/VA

Sphingomonas:4/HV, 2/RA, 1/RN,2/SL, 2/VA

Sphingopyxis: 1/RA

Sphingomonadaceae: 1/RA, 1/RN

Duganella: 2/KK

Herbaspirillum: 1/KK, 2/RN

Actinobacteria: Actinobacteria Bacteroidetes: Flavobacteria Firmicutes: Bacilli

Proteobacteria: Alphaproteobacteria Betaproteobacteria Gammaproteobacteria

31

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-, Beta- and 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 diffi culties in fi nding 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 fl uid 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

32

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 infl ammations 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

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 non-cholera 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

33

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 unidentifi ed 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 endocrine-disruptive 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.

34

Discussion

5.2 Drinking water treatment effi ciencyThe 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-fi ltration 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 fi ltration 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 effi cient in the removal of microcystins. In contrast, it only slightly reduced the endotoxin amounts and was inefficient against the heterotrophic bacteria. The ineffi ciency 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

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 fi laments 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 fi lters, 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

35

Discussion

concentrations in the treated water might be reduced by covering the slow sand fi lter 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 fi ndings indicate the ability of Alphaproteobacteria to survive in water supply systems. Microbes, including bacterial genera such as Pseudomonas and Mycobacterium, produce biofi lms 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 fl ows. Therefore, detected viable heterotrophic bacteria may cause problems within water distribution systems.

36

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

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 unidentifi ed 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

37

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.

38

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 fi nancial 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 scientifi c 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

39

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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.

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 Reason-er’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.

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Actinobacteria; ActinobacteriaClassifi ed at genus level

JO92 FW CYA 872 AM988866 ArthrobacterRU7 FW CYA 791 AM988867 KocuriaET4 FW CYA 838 AM988868 MicrobacteriumHE90 FW BA 830 AM988870 MicrococcusHE95 FW BA 832 AM988871 MicrococcusLO10 FW CYA 740 AM988872 MicrococcusES1 TW R2A 836 AM988869 MicrococcusRA1 TW R2A 833 AM988873 MicrococcusTA6A FW CYA 833 AM988874 MycobacteriumES10 TW R2A 829 AM988875 MycobacteriumKK5 TW R2A 833 AM988876 NocardiaLO12 FW CYA 778 AM988877 NocardioidesJO69 FW Z8 868 AM988878 RhodococcusJO70 FW Z8 868 AM988879 RhodococcusJO72 FW Z8 868 AM988880 RhodococcusJO75 FW Z8 868 AM988881 RhodococcusEN4 FW CYA 741 AM988882 StreptomycesET11 FW CYA 889 AM988883 StreptomycesLO2 FW CYA 823 AM988885 StreptomycesLO4 FW CYA 734 AM988886 StreptomycesLO5 FW CYA 826 AM988887 StreptomycesLO6 FW CYA 819 AM988888 Streptomyces

Appendix 1

53

LO13 FW CYA 701 AM988889 StreptomycesLO14 FW CYA 823 AM988890 StreptomycesPU7 FW CYA 821 AM988891 StreptomycesTE6 FW CYA 830 AM988892 StreptomycesTE13 FW CYA 831 AM988893 StreptomycesTE14 FW CYA 869 AM988894 StreptomycesJO77 FW R2A 869 AM988884 StreptomycesTE37 FW TOX 825 AM988895 StreptomycesTE41 FW TOX 833 AM988896 Streptomyces

Classifi ed at family levelRU14 FW CYA 829 AM988897 Microbacteriaceae

Bacteroidetes; FlavobacteriaClassifi ed at genus level

HE85 FW BA 839 AM988898 ChryseobacteriumHE92 FW BA 849 AM988899 ChryseobacteriumAE103 FW SAA 456 EU724056 ChryseobacteriumAE107 FW SAA 456 EU724051 ChryseobacteriumAE109 FW SAA 456 EU724052 ChryseobacteriumAE110 FW SAA 456 EU724053 ChryseobacteriumAE118 FW SAA 456 EU724054 ChryseobacteriumAE120 FW SAA 456 EU724055 ChryseobacteriumJO2 FW TOX 881 AM988900 ChryseobacteriumJO7 FW TOX 882 AM988901 ChryseobacteriumVA6 TW R2A 843 AM988902 ChryseobacteriumPU5 FW CYA 835 AM988924 FlavobacteriumKU12 FW R2A 841 AM988923 FlavobacteriumRU8 FW R2A 838 AM988927 FlavobacteriumRU9 FW R2A 831 AM988928 FlavobacteriumHA4 FW TOX 837 AM988903 FlavobacteriumJO5 FW TOX 838 AM988904 FlavobacteriumJO9 FW TOX 851 AM988905 FlavobacteriumJO11 FW TOX 791 AM988906 FlavobacteriumJO12 FW TOX 821 AM988907 FlavobacteriumJO15 FW TOX 841 AM988908 FlavobacteriumJO16 FW TOX 879 AM988909 FlavobacteriumJO23 FW TOX 834 AM988910 FlavobacteriumJO24 FW TOX 878 AM988911 FlavobacteriumJO25 FW TOX 838 AM988912 FlavobacteriumJO26 FW TOX 840 AM988913 FlavobacteriumJO27 FW TOX 882 AM988914 FlavobacteriumJO28 FW TOX 835 AM988915 FlavobacteriumJO31 FW TOX 879 AM988916 FlavobacteriumJO32 FW TOX 889 AM988917 FlavobacteriumJO36 FW TOX 838 AM988918 FlavobacteriumJO41 FW TOX 879 AM988919 FlavobacteriumJO104 FW TOX 878 AM988920 FlavobacteriumRJ1 FW TOX 832 AM988925 FlavobacteriumRJ5 FW TOX 834 AM988926 FlavobacteriumTE19 FW TOX 827 AM988929 FlavobacteriumTE25 FW TOX 826 AM988930 FlavobacteriumTE26 FW TOX 841 AM988931 Flavobacterium

Appendix 1

54

TE28 FW TOX 840 AM988932 FlavobacteriumTE31 FW TOX 835 AM988933 FlavobacteriumTE40 FW TOX 824 AM988934 FlavobacteriumKK2 TW R2A 841 AM988921 FlavobacteriumKK3 TW R2A 840 AM988922 FlavobacteriumTE18 FW TOX 845 AM988935 Riemerella

Classifi ed at family levelHE91 FW BA 870 AM988936 Flavobacteriaceae

Bacteroidetes; SphingobacteriaClassifi ed at genus level

OT2 FW CYA 792 AM988938 ArcicellaTE7 FW CYA 831 AM988939 ArcicellaJO107 FW TOX 869 AM988937 ArcicellaTE29 FW TOX 803 AM988940 ArcicellaRU12 FW CYA 842 AM988943 ChitinophagaJO14 FW TOX 888 AM988941 ChitinophagaJO108 FW TOX 884 AM988942 ChitinophagaLI1 FW CYA 809 AM988944 FlectobacillusJO45 FW CYA 840 AM988947 PedobacterPU10 FW CYA 837 AM988949 PedobacterTA2 FW CYA 837 AM988951 PedobacterTE12 FW CYA 884 AM988953 PedobacterIJ6 FW TOX 781 AM988945 PedobacterJO4 FW TOX 884 AM988946 PedobacterPU13 FW TOX 794 AM988950 PedobacterTE5 FW TOX 886 AM988952 PedobacterTE20 FW TOX 833 AM988954 PedobacterTE20B FW TOX 837 AM988955 PedobacterTE23 FW TOX 839 AM988956 PedobacterJO56 FW Z8 882 AM988948 PedobacterJO115 FW TOX 862 AM988957 Spirosoma

Deinococcus-Thermus; DeinococciClassifi ed at genus level

RU1 FW TOX 778 AM988958 DeinococcusFirmicutes; BacilliClassifi ed at genus level

HE105 BW BA 496 AM988965 Bacillus4285C FW BA 496 AM988959 Bacillus4414 FW BA 496 AM988960 BacillusHE99 FW BA 496 AM988964 BacillusET1 FW R2A 858 AM988963 BacillusRJ4 FW TOX 816 AM988966 BacillusTE36 FW TOX 903 AM988968 BacillusES2 TW R2A 863 AM988961 BacillusES8 TW R2A 859 AM988962 BacillusRN13 TW R2A 849 AM988967 Bacillus4361A BW BA 495 AM988969 CaryophanonHE53 FW BA 861 AM988970 LactococcusES4 TW R2A 833 AM988971 Paenibacillus4337D FW BA 497 AM988972 Staphylococcus

Appendix 1

55

4346A FW BA 497 AM988973 StaphylococcusJO48 FW CYA 903 AM988975 StaphylococcusSL5 TW R2A 844 AM988974 Staphylococcus

Proteobacteria; AlphaproteobacteriaClassifi ed at genus level

KU2 FW R2A 805 AM988977 AzospirillumTE21 FW TOX 781 AM988978 AzospirillumJO50 FW Z8 836 AM988976 AzospirillumRN8 TW R2A 840 AM988979 BlastomonasRN9 TW R2A 744 AM988980 BlastomonasHA3 FW CYA 779 AM988981 BoseaLO7 FW CYA 790 AM988983 BoseaRU13 FW CYA 774 AM988986 BoseaTE8 FW CYA 797 AM988987 BoseaTE10 FW CYA 795 AM988988 BoseaTE11 FW CYA 800 AM988989 BoseaTE34 FW TOX 795 AM988990 BoseaKK9 TW R2A 791 AM988982 BoseaRA3 TW R2A 799 AM988984 BoseaRN17 TW R2A 794 AM988985 Bosea4363A FW BA 442 AM988991 BrevundimonasJO43 FW CYA 837 AM988994 BrevundimonasJO46 FW CYA 839 AM988995 BrevundimonasPU6 FW CYA 790 AM989000 BrevundimonasRU6 FW CYA 808 AM989002 BrevundimonasKU11 FW R2A 779 AM988999 BrevundimonasJO6 FW TOX 836 AM988993 BrevundimonasJO103 FW TOX 837 AM988997 BrevundimonasJO62 FW Z8 840 AM988996 BrevundimonasHV3 TW R2A 838 AM988992 BrevundimonasKK4 TW R2A 780 AM988998 BrevundimonasRA10 TW R2A 771 AM989001 BrevundimonasSL1 TW R2A 778 AM989003 BrevundimonasSL2A TW R2A 779 AM989004 BrevundimonasSL2B TW R2A 779 AM989005 BrevundimonasVA7 TW R2A 836 AM989006 BrevundimonasET2 FW CYA 793 AM989007 CaulobacterJO49 FW CYA 838 AM989008 CaulobacterJO91 FW CYA 839 AM989011 CaulobacterJO95 FW CYA 837 AM989012 CaulobacterPU8 FW CYA 799 AM989014 CaulobacterTE1 FW CYA 803 AM989015 CaulobacterJO76 FW R2A 840 AM989010 CaulobacterJO100 FW TOX 840 AM989013 CaulobacterJO51 FW Z8 838 AM989009 CaulobacterEN3 FW CYA 787 AM989016 MethylobacteriumEN5 FW CYA 790 AM989017 MethylobacteriumET7 FW CYA 750 AM989022 MethylobacteriumET10 FW CYA 849 AM989023 MethylobacteriumLO11 FW CYA 788 AM989025 MethylobacteriumOT1 FW CYA 792 AM989026 Methylobacterium

Appendix 1

56

OT4 FW CYA 771 AM989027 MethylobacteriumOT5 FW CYA 742 AM989028 MethylobacteriumOT7 FW CYA 750 AM989029 MethylobacteriumPU2 FW CYA 757 AM989030 MethylobacteriumPU3 FW CYA 757 AM989031 MethylobacteriumTA3 FW CYA 737 AM989032 MethylobacteriumTA5 FW CYA 736 AM989033 MethylobacteriumKU9 FW R2A 875 AM989024 MethylobacteriumES3 TW R2A 794 AM989018 MethylobacteriumES5 TW R2A 799 AM989019 MethylobacteriumES6 TW R2A 800 AM989020 MethylobacteriumES11 TW R2A 799 AM989021 MethylobacteriumET5 FW CYA 795 AM989034 NovosphingobiumTA4A FW CYA 736 AM989036 NovosphingobiumTA1 FW R2A 788 AM989035 NovosphingobiumLO3 FW CYA 809 AM989037 ParacoccusHV6 TW R2A 837 AM989038 PhyllobacteriumHV9 TW R2A 776 AM989039 PhyllobacteriumVA5 TW R2A 835 AM989040 PhyllobacteriumKU5 BW R2A 748 AM989041 PorphyrobacterIJ2 FW CYA 792 AM989042 SphingobiumIJ5 FW TOX 802 AM989043 SphingobiumET6 FW CYA 750 AM989044 SphingomonasJO47 FW CYA 840 AM989051 SphingomonasJO78 FW CYA 839 AM989052 SphingomonasJO79 FW CYA 792 AM989053 SphingomonasJO89 FW CYA 794 AM989054 SphingomonasLI2 FW CYA 840 AM989055 SphingomonasLO8 FW CYA 805 AM989056 SphingomonasTA2A FW CYA 788 AM989062 SphingomonasTU6A FW CYA 798 AM989064 SphingomonasTU3 FW R2A 788 AM989063 SphingomonasJO3 FW TOX 840 AM989049 SphingomonasJO8 FW TOX 817 AM989050 SphingomonasHV2 TW R2A 841 AM989045 SphingomonasHV4 TW R2A 786 AM989046 SphingomonasHV7 TW R2A 832 AM989047 SphingomonasHV10 TW R2A 839 AM989048 SphingomonasRA5 TW R2A 788 AM989057 SphingomonasRA7 TW R2A 789 AM989058 SphingomonasRN2 TW R2A 788 AM989059 SphingomonasSL7 TW R2A 788 AM989060 SphingomonasSL9 TW R2A 803 AM989061 SphingomonasVA4 TW R2A 796 AM989065 SphingomonasVA8 TW R2A 841 AM989066 SphingomonasKU13 FW R2A 787 AM989068 SphingopyxisJO106 FW TOX 841 AM989067 SphingopyxisRA4 TW R2A 789 AM989069 Sphingopyxis

Classifi ed at family levelTA7 FW CYA 803 AM989070 AcetobacteraceaeTA7A FW CYA 788 AM989073 SphingomonadaceaeRA2 TW R2A 788 AM989071 SphingomonadaceaeRN19 TW R2A 842 AM989072 Sphingomonadaceae

Appendix 1

57

Classifi ed at class levelKU10 FW R2A 793 AM989074 AlphaproteobacteriaTA3A FW CYA 800 AM989075 Alphaproteobacteria

Proteobacteria; BetaproteobacteriaClassifi ed at species level

15Ba TOX 1422 AY515379 Paucibacter toxinivorans2a TOX 1423 AY515380 Paucibacter toxinivorans2B15a TOX 1431 AY515381 Paucibacter toxinivorans2B17a TOX 1428 AY515382 Paucibacter toxinivorans2B3a TOX 1421 AY515383 Paucibacter toxinivoransS1030 a,b TOX 1428 AY515384 Paucibacter toxinivoransG44a TOX 1434 AY515385 Paucibacter toxinivoransF39a TOX 1425 AY515386 Paucibacter toxinivoransF36 a,b TOX 1430 AY515387 Paucibacter toxinivorans7Aa TOX 1414 AY515388 Paucibacter toxinivorans2C23a,b TOX 1444 AY515389 Paucibacter toxinivorans2C20T a,b TOX 1429 AY515390 Paucibacter toxinivorans2B5a TOX 1384 AY515391 Paucibacter toxinivorans

Classifi ed at genus levelKU6 BW R2A 837 AM989076 AquabacteriumHE147 FW BA 483 AM989077 Aquitalea4398A BW BA 413 AM989078 BurkholderiaTA13 FW TOX 886 AM989079 Chitinimonas4288A FW BA 483 AM989080 Chromobacterium4336A FW BA 483 AM989081 ChromobacteriumHE106 FW BA 483 AM989082 ChromobacteriumHE127 FW BA 474 AM989083 ChromobacteriumHE160 FW BA 485 AM989084 ChromobacteriumHA1 FW CYA 850 AM989085 DuganellaLO1 FW CYA 839 AM989090 DuganellaTA4 FW CYA 903 AM989091 DuganellaTA11 FW CYA 848 AM989092 DuganellaJO22 FW TOX 807 AM989086 DuganellaJO73 FW TOX 871 AM989087 DuganellaKK1 TW R2A 823 AM989088 DuganellaKK11 TW R2A 823 AM989089 DuganellaEN2 FW CYA 851 AM989093 HerbaspirillumJO80 FW CYA 891 AM989096 HerbaspirillumJO83 FW CYA 893 AM989097 HerbaspirillumLO9 FW CYA 846 AM989100 HerbaspirillumPU4 FW CYA 843 AM989101 HerbaspirillumHA5 FW TOX 848 AM989094 HerbaspirillumJO114 FW TOX 888 AM989098 HerbaspirillumTE24 FW TOX 848 AM989102 HerbaspirillumJO59 FW Z8 855 AM989095 HerbaspirillumKK10 TW R2A 839 AM989099 HerbaspirillumRN14 TW R2A 693 HerbaspirillumRN18 TW R2A 695 HerbaspirillumPU12 FW TOX 802 AM989103 IodobacterJO1 FW TOX 890 AM989104 Janthinobacterium

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58

ET8 FW CYA 847 AM989105 PelomonasET9 FW CYA 886 AM989106 PelomonasHA2 FW CYA 833 AM989107 PelomonasJO84 FW CYA 830 AM989108 PelomonasJO86 FW CYA 835 AM989109 PelomonasJO99 FW CYA 880 AM989110 PelomonasTE4 FW TOX 843 AM989111 RhodoferaxIJ3 FW CYA 839 AM989112 RoseatelesJO87 FW CYA 832 AM989113 RoseatelesJO90 FW CYA 881 AM989114 RoseatelesJO96 FW CYA 829 AM989115 RoseatelesJO97 FW CYA 854 AM989116 RoseatelesKU4 FW R2A 835 AM989117 RoseatelesKU7 FW R2A 817 AM989118 RoseatelesTE15 FW TOX 832 AM989119 VogesellaTE17 FW TOX 849 AM989120 VogesellaTE22 FW TOX 851 AM989121 Vogesella

Classifi ed at family levelKU8 FW R2A 838 AM989122 AlcaligenaceaeJO111 FW TOX 880 AM989123 Incertae sedis 5KU1 FW R2A 851 AM989128 OxalobacteraceaeJO21 FW TOX 797 AM989124 OxalobacteraceaeJO113 FW TOX 891 AM989126 OxalobacteraceaeJO53 FW Z8 889 AM989125 OxalobacteraceaeKK7 TW R2A 847 AM989127 OxalobacteraceaeRN12 TW R2A 806 AM989129 Oxalobacteraceae

Classifi ed at class levelKU14 FW R2A 805 AM989130 Betaproteobacteria

Proteobacteria; GammaproteobacteriaClassifi ed at genus level

4286C FW BA 485 AM989131 Acinetobacter4300B FW BA 485 AM989132 Acinetobacter4362A FW BA 483 AM989133 Acinetobacter4367I FW BA 485 AM989134 AcinetobacterHE21 FW BA 856 AM989137 AcinetobacterHE55 FW BA 853 AM989138 AcinetobacterHE120 FW BA 485 AM989139 AcinetobacterHE124 FW BA 485 AM989140 AcinetobacterHE128 FW BA 485 AM989141 AcinetobacterHE138 FW BA 488 AM989142 AcinetobacterHE139 FW BA 483 AM989143 AcinetobacterHE140 FW BA 483 AM989144 AcinetobacterHE144 FW BA 487 AM989145 AcinetobacterHE145 FW BA 487 AM989146 AcinetobacterHE146 FW BA 487 AM989147 AcinetobacterAU2 FW R2A 849 AM989135 AcinetobacterAU3 FW R2A 847 AM989136 AcinetobacterJO101 FW TOX 895 AM989148 AcinetobacterRU5 FW TOX 853 AM989149 AcinetobacterTE16 FW TOX 850 AM989151 AcinetobacterSL10 TW R2A 851 AM989150 Acinetobacter

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4218B BW BA 492 AM989159 Aeromonas4218E BW BA 492 AM989160 Aeromonas4282D BW BA 492 AM989161 Aeromonas4282J BW BA 492 AM989162 Aeromonas4294H BW BA 492 AM989166 Aeromonas4295A BW BA 492 AM989167 Aeromonas4295F BW BA 491 AM989168 Aeromonas4298D BW BA 491 AM989169 Aeromonas4329A BW BA 492 AM989182 Aeromonas4330A BW BA 489 AM989183 Aeromonas4330D BW BA 492 AM989184 Aeromonas4330E BW BA 492 AM989185 Aeromonas4330G BW BA 494 AM989186 Aeromonas4348A BW BA 491 AM989188 Aeromonas4348D BW BA 494 AM989189 Aeromonas4359B BW BA 492 AM989190 Aeromonas4368D BW BA 492 AM989193 Aeromonas4387A BW BA 492 AM989194 Aeromonas4387B BW BA 492 AM989195 AeromonasHE1 BW BA 867 AM989198 AeromonasHE6 BW BA 861 AM989201 AeromonasHE7 BW BA 864 AM989202 AeromonasHE34 BW BA 857 AM989218 AeromonasHE35 BW BA 857 AM989219 AeromonasHE94 BW BA 856 AM989250 AeromonasHE112 BW BA 492 AM989253 AeromonasHE125 BW BA 492 AM989257 AeromonasHE130 BW BA 489 AM989258 AeromonasHE132 BW BA 489 AM989259 AeromonasHE136 BW BA 492 AM989260 AeromonasHE149 BW BA 492 AM989261 AeromonasHE154 BW BA 492 AM989263 AeromonasHE163 BW BA 492 AM989268 AeromonasAE26 BW SAA 468 EU724006 AeromonasAE27 BW SAA 452 EU724007 AeromonasAE28 BW SAA 468 EU723962 AeromonasAE30 BW SAA 453 EU723973 AeromonasAE31 BW SAA 468 EU723952 AeromonasAE32 BW SAA 444 EU723963 AeromonasAE33 BW SAA 468 EU723902 AeromonasAE34 BW SAA 468 EU723977 AeromonasAE35 BW SAA 468 EU723956 AeromonasAE36 BW SAA 468 EU724009 AeromonasAE37 BW SAA 468 EU724010 AeromonasAE39 BW SAA 468 EU723964 AeromonasAE40 BW SAA 468 EU723967 AeromonasAE41 BW SAA 468 EU723958 AeromonasAE42 BW SAA 454 EU724011 AeromonasAE43 BW SAA 468 EU724012 AeromonasAE44 BW SAA 468 EU723968 AeromonasAE45 BW SAA 468 EU723972 AeromonasAE59 BW SAA 468 EU723978 Aeromonas

Appendix 1

60

AE60 BW SAA 468 EU723979 AeromonasAE61 BW SAA 468 EU724013 AeromonasAE62 BW SAA 468 EU724014 AeromonasAE63 BW SAA 468 EU724015 AeromonasAE64 BW SAA 468 EU723989 AeromonasAE65 BW SAA 468 EU723994 AeromonasAE67 BW SAA 468 EU724016 AeromonasAE72 BW SAA 468 EU724017 AeromonasAE73 BW SAA 468 EU724018 AeromonasAE74 BW SAA 431 EU723912 AeromonasAE81 BW SAA 468 EU724021 AeromonasAE83 BW SAA 468 EU723991 AeromonasAE84 BW SAA 468 EU724022 AeromonasAE85 BW SAA 468 EU723993 AeromonasAE86 BW SAA 468 EU724023 AeromonasAE87 BW SAA 468 EU724024 AeromonasAE88 BW SAA 468 EU724025 AeromonasAE89 BW SAA 468 EU724026 AeromonasAE90 BW SAA 468 EU724027 AeromonasAE91 BW SAA 468 EU723999 AeromonasAE95 BW SAA 468 EU724028 AeromonasAE131 BW SAA 468 EU723948 AeromonasAE137 BW SAA 468 EU723981 AeromonasAE138 BW SAA 468 EU723982 AeromonasAE139 BW SAA 468 EU724045 AeromonasAE141 BW SAA 468 EU724031 AeromonasAE158 BW SAA 468 EU724046 AeromonasAE169 BW SAA 468 EU723965 AeromonasAE170 BW SAA 468 EU723983 AeromonasAE171 BW SAA 468 EU723960 AeromonasAE172 BW SAA 468 EU723974 AeromonasAE174 BW SAA 452 EU723908 AeromonasAE175 BW SAA 468 EU723893 AeromonasAE176 BW SAA 468 EU723900 AeromonasAE188 BW SAA 468 EU723995 AeromonasAE189 BW SAA 468 EU723961 AeromonasAE191 BW SAA 468 EU724035 AeromonasAE192 BW SAA 468 EU723992 AeromonasAE246 BW SAA 431 EU723913 AeromonasAE248 BW SAA 468 EU723996 AeromonasAE249 BW SAA 468 EU724044 AeromonasAE250 BW SAA 468 EU723987 AeromonasAE251 BW SAA 468 EU723997 Aeromonas4192A FW BA 491 AM989152 Aeromonas4201B FW BA 492 AM989153 Aeromonas4201D FW BA 492 AM989154 Aeromonas4201G FW BA 492 AM989155 Aeromonas4207A FW BA 492 AM989156 Aeromonas4207C FW BA 492 AM989157 Aeromonas4207D FW BA 492 AM989158 Aeromonas4286A FW BA 492 AM989163 Aeromonas4286B FW BA 492 AM989164 Aeromonas

Appendix 1

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4287D FW BA 492 AM989165 Aeromonas4300D FW BA 491 AM989170 Aeromonas4300E FW BA 492 AM989171 Aeromonas4301B FW BA 492 AM989172 Aeromonas4301C FW BA 492 AM989173 Aeromonas4301D FW BA 492 AM989174 Aeromonas4302G FW BA 492 AM989175 Aeromonas4316E FW BA 492 AM989176 Aeromonas4316F FW BA 492 AM989177 Aeromonas4318A FW BA 492 AM989178 Aeromonas4318B FW BA 905 AM989179 Aeromonas4318C FW BA 492 AM989180 Aeromonas4326G FW BA 492 AM989181 Aeromonas4337B FW BA 492 AM989187 Aeromonas4365A FW BA 492 AM989191 Aeromonas4366A FW BA 492 AM989192 Aeromonas4400A FW BA 492 AM989196 Aeromonas4402C FW BA 492 AM989197 AeromonasHE3 FW BA 856 AM989199 AeromonasHE4 FW BA 855 AM989200 AeromonasHE9 FW BA 864 AM989203 AeromonasHE10 FW BA 861 AM989204 AeromonasHE11 FW BA 859 AM989205 AeromonasHE12 FW BA 863 AM989206 AeromonasHE13 FW BA 842 AM989207 AeromonasHE14 FW BA 863 AM989208 AeromonasHE15 FW BA 857 AM989209 AeromonasHE16 FW BA 864 AM989210 AeromonasHE17 FW BA 865 AM989211 AeromonasHE18 FW BA 855 AM989212 AeromonasHE19 FW BA 856 AM989213 AeromonasHE20 FW BA 863 AM989214 AeromonasHE22 FW BA 864 AM989215 AeromonasHE28 FW BA 860 AM989216 AeromonasHE29 FW BA 856 AM989217 AeromonasHE37 FW BA 856 AM989220 AeromonasHE38 FW BA 856 AM989221 AeromonasHE39 FW BA 858 AM989222 AeromonasHE40 FW BA 851 AM989223 AeromonasHE44 FW BA 855 AM989224 AeromonasHE45 FW BA 851 AM989225 AeromonasHE46 FW BA 855 AM989226 AeromonasHE47 FW BA 857 AM989227 AeromonasHE48 FW BA 864 AM989228 AeromonasHE51 FW BA 859 AM989229 AeromonasHE56 FW BA 855 AM989230 AeromonasHE57 FW BA 858 AM989231 AeromonasHE60 FW BA 855 AM989232 AeromonasHE61 FW BA 858 AM989233 AeromonasHE62 FW BA 856 AM989234 AeromonasHE63 FW BA 855 AM989235 AeromonasHE64 FW BA 863 AM989236 Aeromonas

Appendix 1

62

HE65 FW BA 864 AM989237 AeromonasHE73 FW BA 859 AM989238 AeromonasHE74 FW BA 866 AM989239 AeromonasHE75 FW BA 856 AM989240 AeromonasHE76 FW BA 859 AM989241 AeromonasHE77 FW BA 858 AM989242 AeromonasHE78 FW BA 856 AM989243 AeromonasHE79 FW BA 857 AM989244 AeromonasHE80 FW BA 863 AM989245 AeromonasHE82 FW BA 864 AM989246 AeromonasHE83 FW BA 858 AM989247 AeromonasHE86 FW BA 858 AM989248 AeromonasHE89 FW BA 858 AM989249 AeromonasHE101 FW BA 491 AM989251 AeromonasHE110 FW BA 492 AM989252 AeromonasHE113 FW BA 492 AM989254 AeromonasHE114 FW BA 491 AM989255 AeromonasHE115 FW BA 492 AM989256 AeromonasHE150 FW BA 492 AM989262 AeromonasHE155 FW BA 492 AM989264 AeromonasHE157 FW BA 492 AM989265 AeromonasHE158 FW BA 492 AM989266 AeromonasHE159 FW BA 492 AM989267 AeromonasHE165 FW BA 492 AM989269 AeromonasTU4 FW CYA 850 AM989271 AeromonasTU6 FW CYA 807 AM989272 AeromonasAE1 FW SAA 481 EU724000 AeromonasAE2 FW SAA 468 EU723949 AeromonasAE3 FW SAA 443 EU723887 AeromonasAE6 FW SAA 468 EU724001 AeromonasAE7 FW SAA 468 EU724048 AeromonasAE9 FW SAA 468 EU723888 AeromonasAE10 FW SAA 468 EU724002 AeromonasAE12 FW SAA 468 EU724003 AeromonasAE13 FW SAA 468 EU724004 AeromonasAE14 FW SAA 468 EU723889 AeromonasAE15 FW SAA 451 EU724008 AeromonasAE16 FW SAA 468 EU723985 AeromonasAE17 FW SAA 443 EU723907 AeromonasAE18 FW SAA 468 EU723953 AeromonasAE19 FW SAA 452 EU724005 AeromonasAE20 FW SAA 468 EU723905 AeromonasAE21 FW SAA 468 EU723998 AeromonasAE23 FW SAA 468 EU723918 AeromonasAE25 FW SAA 468 EU723906 AeromonasAE48 FW SAA 448 EU723947 AeromonasAE53 FW SAA 468 EU723927 AeromonasAE71 FW SAA 468 EU723954 AeromonasAE75 FW SAA 468 EU724019 AeromonasAE78 FW SAA 468 EU724020 AeromonasAE92 FW SAA 468 EU723970 AeromonasAE93 FW SAA 445 EU723915 Aeromonas

Appendix 1

63

AE98 FW SAA 468 EU723932 AeromonasAE99 FW SAA 468 EU723933 AeromonasAE101 FW SAA 468 EU723934 AeromonasAE102 FW SAA 468 EU723935 AeromonasAE104 FW SAA 468 EU723896 AeromonasAE105 FW SAA 468 EU723938 AeromonasAE106 FW SAA 468 EU724029 AeromonasAE111 FW SAA 468 EU724030 AeromonasAE117 FW SAA 452 EU723984 AeromonasAE119 FW SAA 433 EU723916 AeromonasAE121 FW SAA 468 EU723890 AeromonasAE122 FW SAA 468 EU723919 AeromonasAE123 FW SAA 443 EU723895 AeromonasAE125 FW SAA 443 EU723897 AeromonasAE126 FW SAA 443 EU723910 AeromonasAE127 FW SAA 468 EU723898 AeromonasAE128 FW SAA 443 EU723899 AeromonasAE132 FW SAA 443 EU723891 AeromonasAE133 FW SAA 443 EU723892 AeromonasAE143 FW SAA 468 EU723988 AeromonasAE145 FW SAA 468 EU723955 AeromonasAE147 FW SAA 468 EU723959 AeromonasAE150 FW SAA 450 EU723931 AeromonasAE152 FW SAA 468 EU723942 AeromonasAE153 FW SAA 468 EU724032 AeromonasAE154 FW SAA 468 EU723929 AeromonasAE161 FW SAA 468 EU723950 AeromonasAE163 FW SAA 468 EU723951 AeromonasAE165 FW SAA 468 EU723980 AeromonasAE167 FW SAA 467 EU724033 AeromonasAE168 FW SAA 468 EU724034 AeromonasAE180 FW SAA 468 EU723925 AeromonasAE181 FW SAA 468 EU723976 AeromonasAE194 FW SAA 468 EU724036 AeromonasAE195 FW SAA 468 EU723901 AeromonasAE196 FW SAA 468 EU723920 AeromonasAE197 FW SAA 468 EU724037 AeromonasAE198 FW SAA 446 EU723917 AeromonasAE199 FW SAA 468 EU724038 AeromonasAE200 FW SAA 468 EU724039 AeromonasAE201 FW SAA 443 EU723909 AeromonasAE202 FW SAA 468 EU723936 AeromonasAE203 FW SAA 468 EU723969 AeromonasAE204 FW SAA 468 EU723921 AeromonasAE205 FW SAA 468 EU723975 AeromonasAE206 FW SAA 468 EU723894 AeromonasAE207 FW SAA 468 EU723923 AeromonasAE208 FW SAA 468 EU724040 AeromonasAE209 FW SAA 468 EU724041 AeromonasAE210 FW SAA 468 EU723926 AeromonasAE213 FW SAA 468 EU724042 AeromonasAE214 FW SAA 468 EU723914 Aeromonas

Appendix 1

64

AE215 FW SAA 468 EU723928 AeromonasAE216 FW SAA 468 EU723924 AeromonasAE217 FW SAA 445 EU723903 AeromonasAE218 FW SAA 433 EU723930 AeromonasAE219 FW SAA 468 EU723937 AeromonasAE222 FW SAA 468 EU723990 AeromonasAE223 FW SAA 468 EU723940 AeromonasAE230 FW SAA 468 EU724047 AeromonasAE232 FW SAA 468 EU724049 AeromonasAE233 FW SAA 468 EU724043 AeromonasAE234 FW SAA 468 EU723939 AeromonasAE235 FW SAA 445 EU723943 AeromonasAE237 FW SAA 468 EU723922 AeromonasAE238 FW SAA 468 EU723946 AeromonasAE239 FW SAA 468 EU723986 AeromonasAE240 FW SAA 468 EU723945 AeromonasAE243 FW SAA 468 EU723966 AeromonasAE256 FW SAA 443 EU723904 AeromonasAE258 FW SAA 468 EU723971 AeromonasAE259 FW SAA 443 EU723911 AeromonasAE260 FW SAA 468 EU723957 AeromonasJO110 FW TOX 896 AM989270 AeromonasAE162 FW SAA 441 EU724057 Erwinia4329C BW BA 482 AM989273 PseudomonasHE5 BW BA 856 AM989275 PseudomonasHE49 BW BA 851 AM989280 PseudomonasHE54 BW BA 847 AM989281 PseudomonasHE108 BW BA 484 AM989290 PseudomonasHE116 BW BA 482 AM989291 PseudomonasHE2 FW BA 858 AM989274 PseudomonasHE8 FW BA 856 AM989276 PseudomonasHE23 FW BA 857 AM989277 PseudomonasHE24 FW BA 857 AM989278 PseudomonasHE27 FW BA 848 AM989279 PseudomonasHE66 FW BA 850 AM989282 PseudomonasHE67 FW BA 851 AM989283 PseudomonasHE68 FW BA 848 AM989284 PseudomonasHE69 FW BA 849 AM989285 PseudomonasHE70 FW BA 848 AM989286 PseudomonasHE71 FW BA 849 AM989287 PseudomonasHE93 FW BA 847 AM989288 PseudomonasHE103 FW BA 484 AM989289 PseudomonasRU10 FW CYA 813 AM989297 PseudomonasKU3 FW R2A 852 AM989293 PseudomonasJO33 FW TOX 888 AM989292 PseudomonasTE27 FW TOX 848 AM989302 PseudomonasRN1 TW R2A 888 AM989294 PseudomonasRN5 TW R2A 888 AM989295 PseudomonasRN21 TW R2A 889 AM989296 PseudomonasSL3 TW R2A 850 AM989298 PseudomonasSL4 TW R2A 834 AM989299 PseudomonasSL8 TW R2A 852 AM989300 Pseudomonas

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SL11 TW R2A 850 AM989301 PseudomonasVA2 TW R2A 889 AM989303 Pseudomonas4218A BW BA 482 AM989304 Rheinheimera4218G BW BA 483 AM989305 RheinheimeraTE32 FW TOX 838 AM989306 RheinheimeraHE59 FW BA 857 AM989307 SerratiaTE38 FW TOX 855 AM989308 StenotrophomonasHE30 BW BA 855 AM989309 VibrioHE32 BW BA 864 AM989310 VibrioHE33 BW BA 864 AM989311 VibrioHE36 BW BA 855 AM989312 VibrioHE42 BW BA 854 AM989313 VibrioHE43 BW BA 852 AM989314 VibrioHE50 BW BA 854 AM989315 VibrioHE87 BW BA 853 AM989316 VibrioHE88 BW BA 853 AM989317 VibrioHE111 BW BA 487 AM989318 VibrioHE161 BW BA 490 AM989319 VibrioHE162 BW BA 489 AM989320 Vibrio

Classifi ed at family levelHE25 FW BA 854 AM989321 EnterobacteriaceaeHE26 FW BA 855 AM989322 EnterobacteriaceaeHE58 FW BA 860 AM989323 EnterobacteriaceaeHE72 FW BA 852 AM989324 EnterobacteriaceaeAE51 FW SAA 397 EU724058 EnterobacteriaceaeAE164 FW SAA 452 EU724059 Enterobacteriaceae4218F BW BA 492 AM989325 Vibrionaceae

aPaucibacter 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 mi-crocystin-RR. The strains G44 and F36 were shown to degrade also nodularin in preliminary studies.bThe strain was found to degrade microcystins LR and YR as well as nodularin in this study.

Appendix 1