Flow cytometry as a tool to investigate the anoxygenic phototrophic sulfur bacteria coexistence in the chemocline of meromictic Lake Cadagno

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Thesis

Reference

Flow cytometry as a tool to investigate the anoxygenic phototrophic sulfur bacteria coexistence in the chemocline of meromictic Lake

Cadagno

DANZA, Francesco

Abstract

The meromictic Lake Cadagno is characterized by a compact chemocline with high concentration of anoxygenic phototrophic purple sulfur bacteria (PSB) and green sulfur bacteria (GSB). The co-occurrence of PSB and GSB in the same ecological niche makes the chemocline of Lake Cadagno an ideal system for studying the conditions and consequences of coexistence of photosynthetic bacteria population. In this study, flow cytometry (FCM) was applied as a fast tool to identify metabolic changes due to the production and consumption of inclusion bodies and to follow population dynamics of closely related anoxygenic photosynthetic sulfur bacteria in their natural environment. Through this technique the large-celled PSB Chromatium okenii and GSB Chlorobium populations were reliably separated and identified due to difference in auto-fluorescence and cell size. Moreover, we showed that these dominant taxa share the same ecological niche. Two mechanisms for this coexistence were proposed: population activity alternation and bioconvection.

DANZA, Francesco. Flow cytometry as a tool to investigate the anoxygenic phototrophic sulfur bacteria coexistence in the chemocline of meromictic Lake Cadagno. Thèse de doctorat : Univ. Genève, 2018, no. Sc. 5184

DOI : 10.13097/archive-ouverte/unige:103201 URN : urn:nbn:ch:unige-1032010

Available at:

http://archive-ouverte.unige.ch/unige:103201

Disclaimer: layout of this document may differ from the published version.

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Département de botanique et biologie végétale Prof. Dr. Michael Hothorn

Unité de microbiologie Prof. HAS Dr. Mauro Tonolla

Flow Cytometry as a Tool to Investigate the Anoxygenic Phototrophic Sulfur Bacteria Coexistence in the Chemocline of

Meromictic Lake Cadagno

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Francesco Danza de Bellinzona (TI)

Thèse N° 5184

GENÈVE

Atelier de reprographie ReproMail 2018

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ACKNOWLEDGMENTS

First of all, I would like to thank the members of the Jury: Professor Dr. Michael Hothorn, Professor UAS Dr. Mauro Tonolla, Professor Dr. Michel P. Goldschmidt-Clermont, Dr. Jakob Zopfi, and Dr. Frederik Hammes for having accepted to read and valuate this work.

For offering me the opportunity of being part of his research group, for his guidance throughout the time spent at the Laboratorio Microbiologia Applicata of SUPSI Bellinzona and at the Centro Biologia Alpina, I gratefully thank my advisor, Prof. Dr. Mauro Tonolla.

I would like to thank all members of Laboratorio Microbiologia Applicata, and especially the Cadagno team. Thanks to Samuel Lüdin for helpful discussions and Samuele Roman for helping me during lab and field experiment. A big thank you goes to Dr. Nicola Storelli who supported me during these years and especially during the last months.

I thank the personnel of Piora Centro Biologia Alpina, especially Prof. Dr. Raffaele Peduzzi, for laboratory facilities and housing.

Special thank to Dr. Andreas Brüder for helpful discussions during the writing work, and thank you also to Dr. Matthieu Bueche for his kindly collaboration during summers in Cadagno.

Thanks to Eliana, Luca, Daniele, Mahmut, and all my friends for the good times.

I would like to address the last acknowledgement, the most special one, to my family for all their encouragements and their love.

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RÉSUMÉ

Le lac méromictique de Cadagno est situé à 1921 m d’altitude dans la partie sud des alpes Suisses. La minéralogie de son bassin versant est constituée majoritairement de gneiss. La présence d’une veine de dolomie, riche en gypse, a cependant de fortes répercussions sur la chimie des eaux du lac, rendant possible la formation d’une chemocline permanente à cause de la différence de densité entre les eaux profondes et celles de surface. Alors qu’en profondeur, des sources sous-lacustres alimentent le monimolimnion avec de l’eau chargée en sels minéraux, le mixolimnion, est quant à lui alimenté par de l’eau de densité plus faible, pauvre en éléments dissouts. À l’interface entre les deux masses d’eau, un important gradient de sulfures permet d’offrir les conditions nécessaires à la croissance de grandes populations de bactéries phototrophes anoxygéniques sulfureuses. Des bactéries pourpres sulfureuses (PSB, famille des Chromatiaceae) et des bactéries vertes sulfureuses (GSB, famille des Chlorobiaceae), colonisent notamment cet environnement.

Bien que leurs métabolismes soient similaires, ces deux familles de bactéries phototrophes anoxygéniques sont capables de coexister dans la chemocline. Leurs populations sont cependant influencées par les autres organismes vivant dans les compartiment oxiques et anoxiques adjacents. Lors d’un pyrosequençage de la petite sous-unité ribosomique (16S rRNA), il a été observé que les Proteobacteria, les Chlorobi, les Verrucomicrobia et les Actinobacteria étaient les phyla bactériens dominants dans le lac. Pour le mixolimnion, ce sont les Verrucomicrobia et les Proteobacteria qui dominent avec respectivement 46 et 23% d’abondances relatives dans la communauté. La chemocline est quant à elle dominée par les Proteobacteria (84%), alors que le monimolimnion est dominé à 41 et 32% par les Chlorobi et les Proteobacteria. Au cours de la saison estivale, une dynamique de succession entre les populations de Chromatium okenii (PSB, cellules de grandes tailles) et celles d’autres PSB et GSB de plus petites tailles, a été observé lors

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d’une analyse détaillée des phototrophes sulfureuse de la chemocline en utilisant la méthode FISH (fluorescent in situ hybridization). La dynamique de cette succession est clairement soumise à des variations de facteurs environnementaux, mais également à des interactions intra- et interspécifiques.

Dans le cadre de ce projet, des méthodes de cytométrie de flux (FCM) ont été développées et utilisées pour identifier les différentes sous-populations d’organismes phototrophes en utilisant l’autofluorescence émise par leurs pigments photosynthétiques. L’utilisation intensive de la FCM a en outre permit d’évaluer l’activité cellulaire, mais également d’observer les changements métaboliques apparaissant chez les PSB et les GSB lors de situations expérimentales artificielles, mais également en conditions naturelles. Le travail que j’ai effectué a permis de mettre en évidence que l’oxydation photosynthétique des sulfures chez les PSB est corrélée avec des variations de réfraction (sideward scatter - SSC) mesurés en FCM, indiquant des changements au niveau de la complexité cellulaire. La formation et à la consommation d’inclusions intracellulaires, comme les globules de soufre élémentaire (SGBs), participent à ce phénomène.

La FCM permet en outre de faire la distinction entre les C. okenii et les Chlorobium spp. présents dans des échantillons naturels, comme l’eau du lac de Cadagno. La co-occurrence de ces deux taxa tout au long de la saison a ainsi pu être mise en évidence, démontrant par la même leur appartenance à une niche écologique commune. De plus, un phénomène d’alternance temporelle au niveau de la complexité cellulaire des PSB et des GSB, mesuré par FCM, permet d’alimenter la thèse d’une interaction interspécifique dynamique entre ces deux taxa.

Une mesure des taux d’oxydation des sulfures dans des cultures d’enrichissement de C.

okenii, Candidatus “Thiodictyon syntrophicum” Cad16^T, et C. clathratiforme, isolées à partir de l’eau du lac de Cadagno, a démontré que le métabolisme du soufre chez ces espèces était fortement lié aux variations d’intensités lumineuses. Cette découverte permet d’entrevoir les implications environnementales importantes que peuvent avoir de petites variations au niveau de

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la colonne d’eau du lac, en particulier sur l’oxydation des sulfures effectuée par la communauté phototrophique sulfureuse.

Finalement les méthodes développées pour la FCM ont permis une quantification à haute résolution spatiale et temporelle de la présence de C. okenii dans la chemocline. Des mesures de FCM effectuées pendant trois étés consécutifs ont montrées que C. okenii contribuait activement au mélange d’une colonne d’eau d’une épaisseur allant de 0.3 à 1.2 m, phénomène mieux connu sous le nom de bioconvection. Ce travail constitue donc la toute première observation in-situ de ce phénomène dans un environnement naturel. En août 2014 et juillet 2016, la concentration moyenne de C. okenii était de 10⁵ cellules mL^-1, représentant jusqu’à 12% du nombre total de cellules dans la chemocline. Durant ces périodes, la bioconvection particulièrement intense, était en adéquation avec une épaisseur de la couche de mélange plus importante. La dominance saisonnière de C. okenii par rapport aux autre PSB et GSB de plus petites tailles, ainsi que le phénomène de bioconvection que cela engendre, permet d’envisager un bénéfice et un avantage compétitif pour C. okenii. Ce sujet nécessite cependant de plus amples investigations et devra faire l’objet d’études détaillées sur les conséquences que la bioconvection peut avoir sur les conditions microenvironnementales ainsi que les autres membres de la communauté microbienne.

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SUMMARY

Lake Cadagno is a meromictic lake located at 1921 m.a.s.l in the southern Swiss Alps. Its catchment is strongly dominated by gneissic bedrock but contains a dolomite vein rich in gypsum, which strongly influences the water chemistry of the lake. A permanent chemocline has formed between 10 and 14 meters depth as a consequence of the density differences of salt-rich water constantly supplied by underwater springs to the monimolimnion and of electrolyte-poor, low density surface water that feeds the mixolimnion. Steep sulfide gradients in the chemocline support the growth of large populations of anoxygenic phototrophic sulfur bacteria, including purple sulfur bacteria (PSB, family Chromatiaceae) and green sulfur bacteria (GSB, family Chlorobiaceae).

These phylogenetically distinct but metabolically similar anoxygenic phototrophic microorganisms coexist in the chemocline of Lake Cadagno, and are influenced by other organisms in the adjacent oxic and anoxic compartments. High-throughput 16S rRNA pyrosequencing has shown that Proteobacteria, Chlorobi, Verrucomicrobia, and Actinobacteria were the dominant bacterial groups in the lake. In the mixolimnion, Verrucomicrobia and Proteobacteria are the major bacterial phyla with 46% and 23% relative abundance, respectively.

Proteobacteria also dominate the chemocline with 84% relative abundance, whereas in the monimolimnion, Chlorobi and Proteobacteria are the dominant taxa with relative abundance of 41% and 32%, respectively. Fine-scale analysis of anoxygenic phototrophic sulfur bacteria in the chemocline using fluorescence in situ hybridization (FISH) revealed dynamic relationships between populations of large-celled PSB Chromatium okenii and small-celled PSB and GSB over the summer season. This temporal variation of populations of dominant phototrophic sulfur bacteria indicates influence of environmental factors as well as intra- and interspecific interactions on the community composition.

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Flow cytometry (FCM) was developed and extensively applied for the projets of this work to allow rapid identification of photosynthetic microorganisms based on their autofluorescence due to natural pigmentation. Furthermore, FCM was used to evaluate cellular activity and to determine metabolic changes occurring in PSB and GSB in manipulative experiments and natural situations. My work highlighted that photosynthetic sulfide oxidation in PSB co-occurs with variation in sideward scatter (SSC), indicative of changes in cellular complexity due to formation and consumption of inclusion bodies such as sulfur globules (SGBs). Applying FCM with water samples from the chemocline of Lake Cadagno, C. okenii and Chlorobium spp. were reliably distinguished but shown to be often co-occuring, i.e. confirming that they share their ecological niche over seasonal periods. Taking advantage of FCM detection and in particular of measurements of variation in cellular complexity during phases of photosynthetic activity, I identified an alternation in PSB versus GSB cell complexity, which point at the consequences of dynamic interspecific interactions between these taxa.

Moreover, sulfide oxidation rates measured from enrichments of C. okenii, Candidatus

“Thiodictyon syntrophicum” Cad16^T, and C. clathratiforme isolated from Lake Cadagno showed that sulfide metabolism in these species is highly sensitive to changes in light intensity. These findings may have important environmental implications since small changes in the water table of the lake could influence the sulfide oxidation capacity of the community of phototrophic sulfur community.

Finally, we applied the developed FCM approaches to process samples more efficiently to allow for quantification of C. okenii in the chemocline layer with high spatial and temporal resolution. FCM analysis taken over three summer seasons suggested that motile PSB C. okenii are capable of mixing 0.3 to 1.2 m thick water layers in the chemocline, in a process known as bioconvection. Ours was the first observation of bioconvection in the environment. In August

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C. okenii of the total cell number was up to 12%, bioconvection was more intense and resulted in a thicker mixed layer. Bioconvection generated by C. okenii in combination with its seasonal dominance over other small-celled PSB and GSB, may entail a beneficial effect and competitive advantage for C. okenii. However, the consequences of bioconvection on microenvironmental conditions and other members of the bacterial community need to be elucidated with further studies.

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ABBREVIATIONS

ANAMMOX Anaerobic ammonium oxidation ATP Adenosine-5’-triphosphate BChl Bacteriochlorophyll

CARD-FISH Catalysed-reporter deposition fluorescence in situ hybridization CTD Conductivity, temperature and depth

DAPI 4’,6’-diamino-2-phenylindole FACS Fluorescence activated cell sorting

FCC Flavocytochrome c

FCM Flow cytometry

FISH Fluorescent in situ hybridization

FSC Forward scatter

GSB Green sulfur bacteria m.a.s.l meters above sea level

NAD(P)⁺ Nicotinamide adenine dinucleotide (phosphate-oxidase) oxided NAD(P)H Nicotinamide adenine dinucleotide (phosphate-oxidase) reduced NanoSIMS Nano-scale secondary-ion mass spectrometry

NTU Nephelometric Turbidity Unit ORP Oxidation reduction potential OTUs Operational taxonomic units PacBio Pacific Biosciences

PHA Poly(3-hydroxyalkanoates)

PSB Purple sulfur bacteria

Qiime Quantitative insight into microbial ecology

QS Quorum sensing

SGB Sulfur globule

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SGP Sulfur globule proteins SMRT Single-molecule real-time SQR Sulfide-quinone reductase SRB Sulfate-reducing bacteria

SSC Side scatter

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LIST OF TABLES AND FIGURES

INTRODUCTION

p. 4 Fig. 1 Typical water stratification in a meromictic lake p. 8 Fig. 2 Bathimetric map of the Lake Cadagno

p. 10 Fig. 3 Phylogenetic positions of specific clones from a 16S rRNA gene clone library from the chemocline of Lake Cadagno

p. 17 Fig. 4 Light microscopy image of PSB Chromatium okenii showing intracellular sulfur globules

p. 23 Fig. 5 Flow cytometry histogram counts vs bacteriochlorophyll autofluorescence

CHAPTER 1

P. 35 Table 1 Cy3-labeled oligonucleotide probes used for FISH counting

p. 36 Fig. 1 Physico-chemical and biological profiles of Lake Cadagno water column (12 July 2016)

p. 38 Fig. 2 Relative abundance of bacterial communities at the phylum level in Lake Cadagno

p. 38 Fig. 3 Relative abundance of bacterial communities at the genus level in Lake Cadagno

p. 39 Fig. 4 PSB and GSB FISH quantification in July and October 2016

p. 41-42 Fig. 5 Flow cytometry detection of phototrophic populations in the oxic-anoxic transition of Lake Cadagno

p. 48 SI 1 CHAO1 rarefaction curves

p. 48 SI 2 Turbidity profiles of Lake Cadagno water column (28 August 2017)

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CHAPTER 2

p. 61 Fig. 1 Correlation between SGBs per cell and FCM median SSC for anoxygenic PSB C. okenii.

p. 62 Fig. 2 Flow cytometry dynamic response to sulfide oxidation in anoxygenic photosynthetic sulfur bacteria

p. 63 Fig. 3Nucleic acid intensity variation determined by FCM SYBR green staining in PSB C. okenii

p. 64 Fig. 4 Physico-chemical and biological profiles of Lake Cadagno water column p. 65 Table 1 Comparison between FISH and FCM quantification of PSB C. okenii and

GSB Chlorobium spp. in the chemocline of Lake Cadagno

p. 66 Fig 5 Flow cytometry identification of phototrophic populations in the chemocline of Lake Cadagno

p. 66 Fig. 6 Flow cytometry quantification of phototrophic cells in Lake Cadagno water column

p. 67 Table 2 Seasonal cell density for PSB C. okenii and GSB Chlorobium spp. in the chemocline of Lake Cadagno chemocline determined by FCM

p. 68 Fig. 7 In situ cellular complexity variation of PSB C. okenii and GSB Chlorobium spp

p. 76 SI 1 FISH probes percentage for most relevant small-celled PSB in Lake Cadagno chemocline

p. 76 SI 2 Flow cytometry signatures of pure culture of anoxygenic phototrophic sulfur bacteria

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CHAPTER 3

p. 85 Fig. 1 Physico-chemical profiles of Lake Cadagno water column (October 2016) p. 86 Fig. 2 Flow cytometric microbiological analysis of phototrophic microorganisms

in Lake Cadagno

p. 87 Fig. 3 Effect of sulfide concentration on the rate of sulfide loss for chemocline microbial community

p. 88 Fig. 4 Effect of light intensity on the sulfide oxidation rate of C. okenii

p. 89 Fig. 5 Effect of light intensity on the sulfide oxidation of Candidatus “T.

syntrophicum” Cad16^T and GSB C. clathratiforme

p. 95 Fig. 6 C. okenii median SSC variation and sulfide [mM] in function of time p. 96 Fig. 7 Effect of sulfide concentration on maximal median SSC

p. 99 SI C. okenii FSC and SSC average values and standard daviation for three mesurements

CHAPTER 4

p. 106 Fig. 1 Gating strategy of C. okenii for the flow cytometry analysis

p. 107 Table 1 Biological parameters determined for PSB Chromatium okenii in the chemocline of Lake Cadagno over three consecutive summer seasons

p. 117 SI 1 Turbidity and temperature profiles in anoxic layer of Lake Cadagno (11 July 2017)

DISCUSSION

p.119 Fig. 1 Schematic representation of key concepts investigated in this thesis

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INTRODUCTION

1.1 Microbial ecology of meromictic lakes

With about 4-6 × 10³⁰ prokaryotic cells (1), microorganisms dominate the biosphere, constituting about 60% of the Earth’s biomass (2). With approximately 1.2 × 10²⁹ microbial cells in aquatic environments and 4-5 × 10³⁰cells in terrestrial environments, highly diverse microbial communities are essential components of Earth’s ecosystems and represent a large unexplored reservoir of genetic diversity (3).

Microorganisms are key players in fundamental ecological processes and play major roles in biogeochemical cycles through their metabolic activity, thus recycling elements such as carbon, nitrogen, phosphorous, sulfur and most macro- and micronutrients. In freshwater ecosystems, bacteria regenerate and mobilize nutrients in food webs and drive transformation and cycling of most biologically relevant elements. Although large-scale studies have shown that differences in bacterial community composition among lakes can be quite large, there is evidence that many bacterial groups are freshwater-specific and have a global distribution (4). Understanding what microbial groups are present in a given ecosystem and how they interact with each other, with other organisms and with the environment is fundamental for the understanding of ecosystem dynamics and functioning.

1.1.1 Meromictic lakes as model systems for research in aquatic microbiology

The water column of meromictic lakes is generally characterized by two layers with contrasting physical and chemical conditions (Fig.1): top layer, the mixolimnion, which is usually oxic and characterized by one or more complete circulations of the water during the year and a bottom layer, the monimolimnion, which is usually anoxic and characterized by hydrological stability. These layers are separated by a third narrow layer, i.e. the chemocline,

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which is characterized by steep gradients in physical and chemical conditions. Partial mixing events (“meros”) occur regularly in the mixolimnion, because it is directly exposed to the atmosphere, and meteorological events resulting for instance in thermal stratification during summer and mixing events in the cold season due to wind action when the stratifying effect of the heat content is reduced. In high-altitude and high-latitude lakes, freezing of the lake surface might occur in winter, which alters mixing patterns due to cold surface temperatures and absence of wind action. As the exchange of water between the mixolimnion and monimolimnion is relatively weak, chemical gradients of salinity, dissolved gases (oxygen, hydrogen sulfide, methane and others) and nutrients are conserved at the depth of the chemocline (5).

Fig. 1 Typical stratification in small meromictic lakes with the mixolimnion at the top, the intermediate chemocline and the bottom anaereobic monimolimnion. Each layer harbors adapted microbial communities performing characteristic metabolic functions such as oxygenic photosynthesis and aerobic respiration in the oxic mixolimnion, anoxygenic photosynthesis in the chemocline and anoxygenic respiration in both the chemocline and the mixolimnion (modified from Schlegel H.G. (1992) Allgemeine Mikrobiologie Thieme Verlag. 634 pp)

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The chemical composition of meromictic lake waters varies as it does in lakes in general.

Concentrations of total dissolved substances range from low (< 20 mg L^-1) to high (> 300 mg L^-

1). The pH varies from acidic (< 3) to alkaline (> 10), and the redox conditions range from well- oxygenated (Eh about 600 mV) to strongly reduced (Eh < -100 mV). Redox conditions are typically oxic in mixolimnia and permanently anoxic in monimolimnia, but concentrations of reduced chemical species in the monimolimnia, such as ferrous iron, hydrogen sulfide and ammonia can nevertheless vary over a wide range, depending on local conditions. Since the particular physical and chemical properties of meromictic lakes affect the biology, composition, and food-web structure of their communities, meromictic lakes represent stable and peculiar ecosystems interesting for studies on aquatic microbial ecology.

Meromictic lakes are also ideal model systems for studying biogeochemical processes since carbon, sulfur and nitrogen cycles are often associated and occur near the chemocline (6,7). The gradients of density, salinity and dissolved gases in the chemocline create a vertical small-scale distribution of distinct habitats for specific microbial taxa, which allows studies of the vertical zonation of the biogeochemical cycles (8). Examples of such particular microbial driven biogeochemical cycles include the co-occurrence of denitrification and nitrogen fixation (6), but also photoassimilation of inorganic carbon and dark carbon fixation by phototrophic and chemolithotrophic sulfur organisms (7). Additionally, the physico-chemical gradients and the presence of light in the chemocline may support the development of great blooms of phototrophic sulfur bacteria (9,10).

1.1.2 Microbial diversity in meromictic lakes

Globally 177 meromictic lakes are currently known (11). Studies conducted in different meromictic lakes show that bacterial communities differ substantially between oxic and anoxic layers and that both, bacterial diversity and abundance are greater in anoxic than in oxic waters

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patchiness of microbial niches related to zones of distinct biogeochemical activity, abundance of nutrients, lack of mixing and multistep mineralization processes that sustain a diverse and complex bacterial community (14).

Meromictic lakes are environments where the biology and food-web structure are influenced by the physical and chemical properties. In turn, biological processes may also affect the stability of the meromixis. Most of the biological processes contributing to meromixis are located in the anoxic water. In particular, microbial decomposition of organic matter mineralizes soluble compounds such as bicarbonate, sulfide, and ammonium, and contributes significantly to the high density of the water (15). Decomposition of organic matter may also lead to the development of abundant communities of anaerobic phototrophic bacteria in the chemocline, which may compose important components of the food webs of meromictic lakes. Phototrophic sulfur bacteria are common in the chemocline of many meromictic lakes where they use the available light as energy source to reduce compounds, diffusing from the monimolimnion (e.g.

sulfide), as a source of electrons for photosynthesis (16).

In Lake Rogoznica (Croatia), for instance, anoxygenic phototrophic sulfur bacteria were dominant both at the chemocline and in the hypolimnion during stratified periods. However, after an exceptional mixing event, the community of anoxygenic phototrophic sulfur bacteria disappeared and the anoxic water column was dominated by a bloom of gammaproteobacterial sulfur oxidizers (17). In the iron-rich karstic meromictic Lake La Cruz (Spain), oxygenic unicellular cyanobacteria and anoxygenic sulfur phototrophic bacteria develop and may form dense communities close to the chemocline (18). Genetic analyses have recently allowed more detailed investigations into the diversity of bacterial communities and let to the recognition that meromictic lakes, even if they are located near to one another, may have very distinct composition of bacterial communities as observed for Lake Shira and Shunet in Siberia and Lake Oigon in Mongolia (19). Also using genetic analyses, Baatar and colleagues have demonstrated

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that bacterial communities not only differ among these lakes, but that they also differ between oxic and anoxic layers within the same lake.

Biogeochemical processes are key for explaining both the microbial diversity and the interplay between microbial activities and abiotic physico-chemical characteristics. Oxygenic photosynthesis is generally the principal process of inorganic carbon fixation in oxic waters;

however, in meromictic lakes, carbon fixation may be substantially increased due to anoxygenic photosynthesis and chemolithoautotrophy. In the chemocline, sulfur-oxidizing bacteria including Epsilonproteobacteria can contribute to carbon fixation at depths where sulfide and oxygen co- occur (20). In meromictic lakes with high iron concentrations, photoferrotrophy may also contribute to inorganic carbon fixation in zones where Fe²⁺ and oxygen co-occur, if enough light is available (21). Moreover, ANAMMOX – ANaerobic AMMonium OXidation (22)– driven by obligate anaerobic chemolithoautotrophs may also contribute to inorganic carbon fixation in meromictic lakes.

1.1.3 The meromictic Lake Cadagno

Lake Cadagno is a crenogenic meromictic lake located in the Piora valley in the southern Swiss Alps (46°33’N, 8°43’E) at 1921 m.a.s.l, near the Gotthard Pass in the Canton of Ticino.

The valley is characterized by the presence of a longitudinal vein of dolomite rock, which is rich in gypsum and separates two layers of pre-Triassic gneiss and mica-schists of central alpine crystalline rocks. This particular combination of distinct types of bedrock have created sublacustrine springs in the southern part of Lake Cadagno, with characteristic consequences on the physico-chemistry of its waters (Fig. 2). The lake, 842 m long and 423 m wide with an average surface area of 26 ha and a maximum depth of 21 m (23), is covered by ice and snow for up to 6 months during winter and spring.

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Fig. 2 Bathimetric map of the Lake Cadagno and its hydrogeological context modified from Wirth et al. (24).

Subaquatic springs are shown as white dots and surface in- and out- flows of water are represented by black arrows.

Water that flows through the dolomite bedrock becomes enriched in calcium, magnesium, carbonate and sulfate, and in turn contributes to the stability of the meromixis in the lake.

Consequently, the permanent chemocline is stabilized by the strong density gradients between the two water strata of the upper mixolimnion and the lower monimolimnion (23).

Due to difficulties in culturing environmental microorganisms, the study of bacterial communities in Lake Cadagno began with direct detection methods such as the DAPI staining of nucleic acids. This approach revealed many bacterial morphotypes and allowed first evaluations of the bacterial diversity in the water column of the lake. Amplification and generation of gene clone libraries for 16S ribosomal RNA genes then allowed further analysis of bacterial communities in the chemocline (6,25–27), in the monimolimnion and the mixolimnion (25,28,29).

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These studies have shown that the unusually high concentrations of sulfate and the presence of organic compounds favors the development of sulfate-reducing bacteria (SRB) in the monimolimnion and in the anoxic sediments. Their activity may raise concentrations of hydrogen sulfide ions (HS^-) up to 0.2 to 1 mM in the monimolimnion. In turn, sulfide (H2S) supports the proliferation of a dense community of phototrophic sulfur bacteria in the chemocline at a depth of approximately 12 m, i.e. in a zone where light meets the sulfide front that diffuses from the anoxic layer. By metabolizing reduced sulfur compound such as sulfide and thiosulfate (S2O32-), phototrophic sulfur bacteria produce elemental sulfur (S⁰) first and then sulfate. Indirectly, their activity thus also benefits communities of eukaryotic aerobic organisms in the upper mixolimnion, which would be otherwise be affected by toxic concentrations of these compounds.

During summer, a dense community of anaerobic phototrophic sulfur bacteria of up to 10⁷ cells mL^-1 inhabits the chemocline, composed of photosynthetic purple sulfur bacteria (PSB;

Chromatiaceae) of the genera Chromatium, Lamprocystis, Thiocystis and Thiodictyon and green sulfur bacteria (GSB; Chlorobiaceae) of the genus Chlorobium (25,28,30). The phylogeny and the population dynamics of phototrophic sulfur bacteria in the chemocline have been studied with molecular methods based on 16S rRNA sequencing (25,31,32) (Fig. 3). These studies provided identification of novel bacterial species, which were later also isolated (e.g Thiocystis chemoclinalis sp. nov., Thiocystis cadagnonensis sp. nov. and Candidatus “Thiodictyon synthrophicum” sp. nov.) (33,34).

A recent study focusing on methane oxidation coupled with oxygenic photosynthesis in the chemocline revealed some evidence of methane-oxidizing activity, but failed to detect anaerobic methane-oxidizing archaea (ANME)-1 or (ANME)-2 (29).

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Fig. 3 Neighbor-joining tree depicting the phylogenetic positions of specific clones from a 16S rRNA gene clone library from the chemocline of Lake Cadagno. Adapted from Tonolla et al. (2005) (25).

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1.2 Anoxygenic phototrophic sulfur bacteria

Anoxygenic phototrophic sulfur bacteria are organisms commonly found in illuminated anaerobic aquatic environment containing hydrogen sulfide. They can therefore colonize the top anoxic layers in stratified lakes, and the top layers of anoxic illuminated sediments where they establish microbial mats (16). Correlations between anoxygenic photosynthesis and irradiance suggest that light is the principal physical environmental variable controlling the activity of phototrophic sulfur bacteria (16). Moreover, all anoxygenic phototrophic sulfur bacteria depend on electron donors with standard redox potentials more negative than those of water, which include H2S, H2, or acetate. The permanent stratification of meromictic lakes consequently fosters the formation of communities of phototrophic sulfur bacteria throughout the year.

1.2.1 Sulfur metabolism in anerobic microorganisms

Sulfur is an essential part for various organic compounds including amino acids, polypeptide, enzyme cofactors, antibiotics, and carbohydrates with diverse biological functions (35). Sulfur exists in several stable oxidation states and presents high reactivity in reduced forms. Among the different oxidation states that exist in nature, sulfur is found in significant amounts as sulfide (- 2), elemental sulfur (0) and sulfate (+6). Transformations between these three main states may result from biological and chemical processes. The major forms of sulfur available in nature are sulfate or sulfide in water and soil and sulfur dioxide in the atmosphere.

In absence of oxygen, sulfate (SO42-) is used as a terminal electron acceptor for respiration by sulfate-reducing bacteria (SRB). Lactate, pyruvate, malate, and acetate are used as electron donors and they are oxidized to CO2 at the end of the respiratory pathway. The product of anaerobic respiration of sulfate is sulfide (H2S) which is released into the environment where it contributes to maintain anaerobic conditions. Sulfate- and sulfur-reducing bacteria are metabolically versatile groups of microorganisms that belong to different families and genera.

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bacteria, in the genus Thermodesulfobacterium, and in some Archaea. The incorporated sulfate is first transformed in adenosine-5’-phosphosulfate (APS) by ATP sulfurylase and subsequently converted into 3’-phosphoadenosine-5’-phosphosulfate (PAPS) by APS kinase (36).

Hydrogen sulfide (H2S) is the most abundant reduced form of inorganic sulfur in aquatic environments, and results from the anaerobic respiration of SRB, generally in sediments or in hydrothermal vents. It serves as electron donor in the energy-generating systems of photo- and chemo-lithotrophic bacteria. Reduced sulfur compounds such as hydrogen sulfide play important roles as electron donors in the phototrophic carbon dioxide fixation. During the autotrophic growth of phototrophic sulfur bacteria, sulfide provides electrons to a membrane-bound electron transport system in a process that ultimately leads to the production of reducing power molecules such as NAD(P)H and reduced ferredoxin, which are required for the inorganic carbon fixation.

This transfer of electrons requires energy that is provided by light in phototrophic bacteria (37) and by dark redox reactions of inorganic substrates in chemo-lithotrophic bacteria (38).

1.2.2 Light-dependent CO2 fixation in anoxygenic PSB and GSB

Purple sulfur bacteria (families Chromatiaceae and Ectothiorhdospiraceae) and green sulfur bacteria (phylum Chlorobi) utilize reduced sulfur compounds as electron donors. They couple the oxidation of various sulfur compounds to CO2 reduction according to the following equations:

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CO2 + 2H2S [CH2O] + H2O + 2S⁰ 3CO2 + 2S⁰ + 5H2O 3[CH2O] + 2SO42- + 4H⁺

2CO2 + H2S + 2H2O 2[CH2O] + SO42- + 2H⁺ CO2 + 2S2O32- + H2O [CH2O] + 2S⁰ + 2SO42-

2CO2 + S2O32- + 3H2O 2[CH2O] + 2SO42- + 2H⁺

Besides anoxygenic phototrophic sulfur bacteria, some members of Chloroflexaceae, and the strictly anaerobic Gram-positive heliobacteria are also able to oxidize reduced sulfur compounds during photosynthesis (35).

The molecules of bacterio-chlorophyll (BChl) composing the light-harvesting antennae are different in PSB and GSB (39). The dominant pigments found in PSB are BChl a and b, while in GSB BChl c, d and e are present. Even the properties of BChl structural complexes differ between PSB and GSB. In PSB, two light-harvesting complexes (LH1 and LH2) are observed, whereas in GSB, these complexes are organized into highly structured organelles known as chlorosomes (40) which allow a highly efficient absorption of the energy of light. GSB may thus be better adapted than PSB to grow at low light intensities, and probably for this reason they are normally found in greater depth of lakes and/or just under the PSB layer, if these populations coexist in the same habitat (41).

Pigments in the light-harvesting antenna complexes adsorb light radiation and transfer the captured energy to the photosynthetic reaction center where it is used to generate ATP and reductant NAD(P)H. Photosynthetic reaction centers are divided into two main groups: a type I, present generally in GSB, in which a ferredoxin (Fe-S) cluster acts as the terminal electron, and a type II, present mainly in PSB and other phototrophic bacteria, which uses a quinone as a terminal electron acceptor. Anoxygenic phototrophic sulfur bacteria utilize either the

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pheophytin-quinone reaction centers (PSB and Chloroflexus spp.) or the Fe-S reaction centers (GSB and heliobacteria). The transfer of electrons from the photosynthetic reaction center to the cytochrome complex and to NAD(P)⁺ or other cofactors (e.g. ferredoxin in GSB) establishes a proton-motive force across the membrane, which is used to produce ATP by ATPases.

PSB mainly assimilate CO2 using the Calvin-Benson-Bassham cycle (CBB cycle), in which ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCO) is the key enzyme. Three major steps occur in the CBB cycle: (a) carbon fixation, (b) reduction of the fixed carbon and (c) regeneration of the molecules involved in the cycle. The first reaction, i.e. carbon fixation, is performed by RuBisCO that catalyzes the carboxylation of ribulose-1,5-bisphosphate (RuBP) into two molecules of 3-phosphoglyceric acid (3-PGA) using CO2 as substrate. In the second step, 3-PGA is further metabolized and reduced in triose phosphate using ATP and NAD(P)H.

The CBB cycle produces one molecule of triose phosphate (glyceraldehyde-3-phosphate, GAP) from three molecules of CO2. As a third step, the cycle regenerates RuBP. The reactions of regeneration in the CBB cycle are essentially catalyzed by non-specific enzymes also involved in other metabolic pathways, except for the phosphoribulokinase which regenerates RuBP from ribulose-5-phosphate (Ru-5-P) using one molecule of ATP. Globally, the CBB cycle consumes nine ATP and six NADPH equivalents for the synthesis of one molecule of triose phosphate (42).

The citric acid cycle (TCA), also known as “Krebs cycle”, is a process utilized by aerobic organism to generate energy (ATP) and reducing agents (NADPH) via the oxidation of acetate, derived from carbohydrates, fats, and proteins, to CO2. However, some microorganisms such as GSB use the reductive reverse tricarboxylic acid (rTCA) cycle to assimilate CO2. Several enzymes required for the rTCA are also active in the Krebs cycle, but a number of irreversible reactions of the rTCA cycle need specific enzymes such as a fumarate reductase, a ferredoxin-

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dependent 2-oxoglutarate synthase, and an ATP-citrate lyase. These three enzymes are usually regarded as the characteristic enzymes of the rTCA cycle (42).

1.2.3 Sulfide oxidation in PSB and GSB

The first enzymatic step in sulfide oxidation was initially thought to be due to flavocytochrome c (FCC), which carries out hydrogen sulfide-dependent cytochrome c reduction. However, findings from subsequent studies are, in favor of sulfide-quinone oxidoreductase (SQR), a flavoenzyme belonging to the large disulfide oxidoreductase family (43). During autotrophic growth of bacteria, H2S provides the electrons for the NAD⁺ reduction, required for CO2 fixation. Phototrophic sulfur bacteria use light energy for the upward transport of electrons from sulfide to NAD⁺. The electrons are first transferred from sulfide to quinone and, subsequently, they are transported to NAD⁺by a reverse electron transfer catalyzed by the NADH dehydrogenase. This reverse transport requires an electrochemical proton potential across the cytoplasmic membrane, which is generated by the cyclic electron transport through the photosystem. When electrons enter the electron transport chain via FCC at the level of cytochrome c, they are transported upwards to NAD⁺ by the reverse electron transport through the cytochrome bc complex, the quinone pool and NADH dehydrogenase.

On the other hand, if the electrons enter the electron transport chain at the level of quinone via SQR, the cytochrome bc complex contributes to the generation of an electrochemical proton potential required for the upward transport of the electrons from quinol to NAD⁺ through the NADH dehydrogenase. Sulfide oxidation by SQR therefore provides more energy than sulfide oxidation by FCC (43). SQR is a single polypeptide with a molecular mass of 55 kDa (44). It is a membrane-bound enzyme that belongs to the glutathione reductase family of flavoproteins. SQR activity has been found to be widely distributed among prokaryotes, including PSB and GSB (45).

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1.2.4 Inclusion bodies in phototrophic sulfur bacteria

Inclusions in prokaryotes are considered as localized intracellular accumulations of metabolic products. Inclusions represent a variety of accumulated materials, and are therefore further differentiated into granules, globules, or crystals. Inclusions may be composed by inorganic or organic substances, single elements or compounds like peptides or proteins, carbohydrates or some type of fatty materials, and they may function as a storehouse of these substances (46). The ability to store surplus substrates during unbalanced growth constitutes an advantageous evolutionary strategy that enables microorganisms to cope with fluctuating environmental conditions.

Synthesis and accumulation of storage materials in phototrophic sulfur bacteria occur under diverse environmental conditions (47). Phototrophic sulfur bacteria can accumulate zero-valence sulfur S⁰globules (SGBs), glycogen, poly(3-hydroxyalkanoates) (PHA) and polyphosphate.

These compounds are stored intracellularly except for sulfur which is stored extracellularly in the case of GSB.

Sulfur globules

Phototrophic sulfur bacteria using reduced sulfur compounds during anoxygenic photosynthesis can accumulate SGBs (Fig. 4), which are oxidized to sulfate if reduced sulfur compounds become exhausted. SGBs are formed extracellularly in the case of GSB as well as the PSB members of the family Ectothiorhodospiraceae, while they are formed intracellularly in members of PSB belonging to the Chromatiaceae (47).

SGBs can reach diameters up to 2 µm and appear highly refractile in light, which allows their observation by light microscopy. They are delimited by a protein envelope composed of two major types of “sulfur globule proteins” (SGP) of ca. 10.5 kDa (SgpA and SgpB) and 8.5 kDa (SgpC) (48–50). Deposition of sulfur in globules by cells of Allochromatium vinosum and

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Chromatium warmingii may increase their density from 1.1500 to 1.2281 and from 1.0890 to 1.1321 g cm^-3, respectively (47,51).

In A. vinosum (Chromatiaceae) SGBs are formed from the oxidation of sulfide, thiosulfate, polysulfide and extraneous sulfur. In this species, the formation of SGBs is obligatory during the oxidation of these substrates to the end product sulfate. The main enzymes believed to be involved to catalyze the oxidation of sulfide are the periplasmic FAD- containing flavocytochrome c and the membrane-bound sulfide:quinone oxidoreductase (SQR) (50).

For the degradation of sulfur globules, the only gene region currently known to be essential for oxidation of stored sulfur was localized by interposon mutagenesis in Allochromatium vinosum. In particular, the dsrAB products form the α2β2-structured sulfite reductase, a protein closely related to the dissimilatory sulfite reductases of SRB (50).

Fig. 4 Light microscopy image of PSB Chromatium okenii showing intracellular sulfur globules (adapted from (32))

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Glycogen

Many bacteria accumulate glycogen particularly under excess of carbon and energy during growth. The structure of bacterial glycogen is similar to that observed for mammalian glycogen.

In anoxygenic phototrophic sulfur bacteria, accumulation of storage carbohydrates is observed simultaneously with the production of elemental sulfur during phototrophic growth based on sulfide. Glycogen is a polymer in which numerous glucose residues are joined together by α- (1,4)-glucosidic linkages into long chains which are branched with α-(1,6) linkages at the branch points (47).

Poly(3-hydroxyalkanoates)

A large variety of prokaryotes are capable of accumulating polyhydroxyalkanoates (PHA) as water-insoluble inclusions in the cytoplasm, which are referred to as PHA granules. PHA are accumulated intracellularly by PSB both when growing in the light in the presence of organic acids, and when growing in the dark, as a byproduct of the degradation of intracellularly stored glycogen. PHA are not observed in GSB (47).

Polyphosphate

Polyphosphate are linear polymers of orthophosphate with variable chain length which can be found as a variety of different fractions, forming intracellular granules. However little is known about accumulation of polyphosphate by phototrophic sulfur bacteria (47).

1.2.5 Diversity and temporal dynamic of phototrophic sulfur bacteria in Lake Cadagno In the sulfur rich Lake Cadagno an abundant community of anoxygenic phototrophic sulfur bacteria develops in the chemocline, especially during spring and summer months. Cell concentration maxima of anoxygenic phototrophs Chromatiaceae and Chlorobiaceae in summer occur generally between 10.5 and 13.5 meters depth (26). The main anoxygenic phototrophic bacteria species identified in Lake Cadagno are the flagellated PSB Chromatium okenii (large cell), Thiocystis chemoclinalis and Thiocystis cadagnonenensis (small cells), the immotile small-

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celled PSB Lamprocystis purpurea, Candidatus “Thiodictyon syntrophicum”, Lamprocystis roseopersicina and Lamprocystis spp. strain D, and two GSB Chlorobium clathratiforme and Chlorobium phaeobacteroides (26,31,34,52).

PSB belong to the gamma division of Proteobacteria. Metabolically, PSB can be photoautotrophs or photoheterotrophs. Some species, such as Lamprocystis purpurea and Candidatus “Thiodictyon syntrophicum” can exist as chemolithotrophs in the absence of light (16). PSB are mainly obligate anaerobes although some may tolerate low-oxygen concentrations.

Small-celled PSB are between 1.4 and 4 µm in diameter and have a spherical to oval form. They are immotile and contain gas vacuoles. Instead large-celled PSB, e.g. C. okenii (size: 4.5 - 6 × 8 - 15 µm), are motile due to their flagella. The seasonal distribution of PSB in the chemocline may change during the season and over the years. In 2000, a shift in dominance was observed in the Chromatiaceae community, i.e. changing from a dominance of C. okenii in spring and early summer to an increase in L. purpurea in the late summer and autumn (26). In contrast, C. okenii was observed at high densities in late autumn of the year 1998, whereas small-celled PSB were most abundant during spring and summer (52).

GSB belong to the separate phylum Chlorobi. They are generally considered as strict photoautotrophs, fixing CO2 using the reverse citric acid cycle. The occurrence of the pigments in the concentrated chlorosomes allows GSB to use light at very low intensities (16,41), and therefore they are usually at highest concentration just below the PSB maximum.

At the beginning of the 21th century, in Lake Cadagno the abundance of PSB and GSB has radically changed. Until 2001, PSB were the dominant phototrophic sulfur bacteria, constituting 70-95% of the total bacterial population. In 2000 the dominance shifted from PSB to GSB due to the increase of C. clathratiforme, which thereafter represented up to 95% of the community of phototrophic sulfur bacteria in the chemocline (25).

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1.2.6 Anoxygenic phototrophic sulfur bacteria and primary production in Lake Cadagno The bacterial community in the chemocline of Lake Cadagno may contribute up to half of the total daily carbon photoassimilation (7). Rates of CO2 assimilation of the most abundant phototrophic sulfur bacteria in the chemocline were estimated using nanoscale secondary ion mass spectrometry (nanoSIMS) (53,54) and ¹⁴CO2 assimilation assays in dialysis bags (55). The strongest assimilator in the presence of light was the large-celled PSB C. okenii, whereas in the dark it was the small-celled PSB Candidatus “T. syntrophicum”, which, however, can also show high carbon assimilation rates in the light (55). In these studies, C. okenii represented only 0.3%

of the total abundance, but it contributed 70% to the total carbon uptake in the system. This high assimilation could be due to the ability of C. okenii to optimize its location on the physico- chemical gradients in the chemocline by moving towards high substrate concentrations and optimal light conditions (53). The most abundant population in the chemocline during that study, i.e the GSB C. clathratiforme showed only low carbon assimilation rates (53,55). Micro- autoradiographic analysis revealed that about 10% of the C. clathratiforme cells in the layer below the photic zone fixed carbon in the dark, compared to 20-55% in the photic zone using light (56). Because GSB are considered to be obligate phototrophs, other metabolic pathways probably contribute to its growth.

Although non-photosynthetic carbon assimilation is often observed in phototrophic sulfur bacteria, its metabolic pathway is not yet fully understood. In order to gain insight into the process of dark carbon assimilation, two-dimensional gel electrophoresis was used to monitor the global changes in the proteome of Candidatus “T. syntrophicum” strain Cad16^T in anoxic autotrophic cultures. Interestingly, three enzymes that were more abundant in the dark are part of a hypothetical anaerobic dicarboxylate/4-hydroxybutyrate (DC/HB) cycle. The substrates needed for this process should be provided by the degradation of the storage globules of PHB, whose synthesis was shown to be higher in the presence of light (57).

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1.3 Flow cytometry in aquatic microbial ecology

Flow cytometry (FCM) is used for analyzing and counting particles by suspending them in a flow stream passing through an excitation light source like a laser beam. Interaction between the light beam and the particle causes characteristic scattering of light and the excitation of fluorochromes if present in the particles. The scatter signal is divided in “forward scatter (FSC)”, or small angle signal, measuring light diverted at a low angle (0.5 – 5°), and “sideward scatter (SSC)”, or large angle signal, measuring light collected at angles greater than 15°. The angle of scattered light provides information on the nature of particles including surface and intracellular characteristics. The cell suspension may be stained using fluorophore dyes that can be design to attach to particular molecules such as DNA, RNA, protein, or antibodies or by using fluorescent oligonucleotides probe.

Histograms with two variables (e.g. light scattering versus fluorescence, fluorescence versus counts) or contour plots may be generated from scattered light, which provide information on structural properties of the analyzed cell populations (58,59).

1.3.1 Application of flow cytometry in aquatic microbiology

Already applied for more than 30 years in medical research and routine diagnosis/analysis, FCM has been increasingly used also in the field of aquatic microbiology during the last three decades.

Since less than 1% of microbial species present in the environments are cultivable on solid media, microscopy is the routine technique for detection and quantification of bacteria, especially in aquatic samples. FCM is a powerful tool for counting of microorganisms and in combination with various nucleic acid stains (SYTO 9, SYTO 13, SYBR Green) it has been used for quantification of microbial communities in various aquatic environments (60). FCM procedures have been extensively applied and are now an established and accepted approach for the quantification of total bacterial abundance in drinking water distribution systems and

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wastewater (61–64). Moreover, it has been demonstrated that the total cell number is a good indicator of water quality and that it may change during treatment of waste water usings sand filtration, granular activated carbon, and ozonation. Disinfection treatments of waters using chemical oxidants may differ in selectivity and reactivity and thus the efficiency of bacterial disinfection processes might vary. FCM can be applied to evaluate both the cellular activity and the physiological state of microbial cells. For instance, it allows differentiating between living and dead cells. The combination of propidium iodide (PI) and SYBR Green stains is generally used to monitor membrane integrity and, consequently, cell viability (58,61). In combination with nucleic acid staining FCM can be applied to separately count bacteria from low nucleic acid (LNA) and high nucleic acid (HNA)- containing bacteria. The discrimination between these two groups is based on differences in fluorescence intensity that in turn is related to the nucleic acid content (60,65). Clusters of low-DNA and high-DNA bacteria are widespread in nearly all aquatic samples, from marine, brackish and freshwater environments.

1.3.2 Application flow cytometry for research on phototrophic organisms

The natural cellular pigmentation of phototrophic bacteria can be used as a signal to discriminate phototrophic and heterotrophic microorganisms. Chlorophyll (found naturally in all phytoplankton cells) and phycobilin (found in cyanobacteria) are natural fluorophores showing characteristic optical wavelength excitation and emission profiles. The fluorescence of chlorophyll can thus be used as the primary gating factor to discriminate phytoplankton from other particles. Chlorophyll a is the dominant photosynthetic pigment in most phytoplankton species and its excitation with blue laser light (488-nm) results in a measurable fluorescent signal in the long red wavelength (> 640 nm) (Fig. 5).

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Fig. 5 Natural fluorescence of bacteriochlorophyll in the long red wavelengths (FL3, red filter > 640 nm).

Histogram without phototrophic sulfur microorganisms (left), and after addition of phototrophic sulfur bacteria (right). Threshold for bacteriochlorphyll autofluorescence signal corresponded to FL3 > 1’000. The vertical red line separates the non-auto-fluorescent cells (left, from 10¹ to 10³) from the phototrophic auto-fluorescent cells (right, from 10³ to 10⁷). Note the large difference in y-axis range between the two panels.

Natural fluorophores provide the great advantage in that they allow detection, discrimination, morphological analysis and quantification of microorganisms using FCM without the addition of dyes such as SYBR Green (66). FCM can thus be used to analyze phytoplankton from freshly collected environmental water samples by measuring relative cell size and intrinsic fluorescence profiles (67,68). Other accessory pigments in phytoplankton are the phycobilins and the carotenoids. Phycocyanins are a class of phycobilins present in all species of phototrophic cyanobacteria and have been widely used for detection of cyanobacteria. The red FCM (640 nm) maximally excites phycocyanins near their absorption maximum producing strong fluorescence emissions detected at 675 nm (66).

Due to these developments, FCM has become an established method for phytoplankton studies (67–69). The main advantage of this technique lays in the processing of samples at a high rates, allowing the analysis of a large number of individuals. Moreover, each analyzed cell is

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characterized by several parameters, such as size, light scatter, and fluorescence emission, which allows estimates of the relative abundance of cells belonging to several groups. These approaches were used, for instance, for differentiating photosynthetic from non-photosynthetic prokaryotes, for measuring bacterial cell size and nucleic acid content and for estimating the relative activity and physiological state of each cell (68).

The potential of FCM as a fast tool for the characterization and counting of phototrophic sulfur bacteria has documented in the past. Purple and green sulfur bacteria from both laboratory strains and from environmental samples obtained from the stratified meromictic Lake Vilar (Spain) were detected and counted in unstained samples using a blue laser-based FCM (70).

Moreover, variations in cell-specific pigment content and the dynamics of sulfur accumulation were also quantified using FCM as sulfur accumulation changes the light scatter characteristic of the phototrophic cells. Therefore, the rapid identification and the physiological characterization provided by FCM could be applied in complex ecophysiological experiments in natural environments.

In detailed studies of bacterial populations analysis, FCM combined with flow cell sorting (also known as fluorescence-activated cell sorting – FACS) has been applied to concentrate and count subpopulations from complex bacterial communities (71,72). FCM has also been combined with radioactive or stable isotope incubations of the sorted sub-populations in order to measure the metabolic activity of specific functional groups (73). In a recent study, samples from Lake Cadagno incubated with ¹⁵N2 and ¹³CO2 were flow cell sorted using gating criteria based on the auto-fluorescence signature of the green sulfur bacterium Chlorobium phaeobacteroides. The sorted cells were subsequently transferred to a filter membrane for NanoSIMS analysis (54).

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1.4 Aim of the study

The chemocline of Lake Cadagno is characterized by a dense population of phototrophic sulfur bacteria, dominated by two major populations: i) the large-celled PSB C. okenii, and ii) the GSB Chlorobium spp. (C. clathratiforme and C. phaeobacteroides). In the past, investigations on Lake Cadagno chemocline were principally directed towards phylogenetic studies using FISH, and towards measuring metabolic activities of single populations of phototrophic sulfur bacteria (54–56). From other ecosystem compartments than the chemocline, only few indications concerning the microbial biodiversity are available. Furthermore, the mechanisms of causes of coexistence of different PSB populations in the same habit, as well as that of PSB and GSB remains elusive. Similarly, the consequences of this coexistence on microenvironmental conditions and on the biogeochemistry of the lake is unknown.

The aim of my study was to expand our knowledge on the diversity and functioning of the microbial community in the water column of Lake Cadagno. I aimed at understanding the specific mechanisms and strategies underlying the coexistence of the dominant phototrophic sulfur bacteria C. okenii and Chlorobium spp. and whether this coexistence affects the surrounding environment. For that purpose, I developed and applied flow cytometry (FCM) as an alternative technique for the rapid identification and quantification of photosynthetic microorganisms in Lake Cadagno. Furthermore, I tested the use of FCM as a tool for in situ spatio-temporal analyses and to perform ecophysiological experiments both under laboratory conditions and in the natural environmental conditions.

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Therefore, the following principal investigations are addressed in my thesis:

1. Investigations on microbial biodiversity over the whole water column of Lake Cadagno and on the consequences of microbial interactions within and between different water layers (Chapter 1)

2. Investigations on the activities of anoxygenic phototrophic PSB and GSB both under laboratory and in situ conditions through flow cytometry (Chapter 2 and Chapter 3) 3. Investigations on light-dependent sulfide oxidation of PSB and GSB isolated from Lake

Cadagno (Chapter 3)

4. Investigations on the impact of phototrophic sulfur bacteria on the surrounding environment and in particular on the stability of the stratification of Lake Cadagno (Chapter 4)

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CHAPTER 1

Phylogenetic diversity of the microbial community in the water column of meromictic Lake Cadagno and evidence for seasonal dynamics

Francesco Danza^1,2, Damiana Ravasi¹, Nicola Storelli¹, Samuele Roman^1,3, Samuel Lüdin^1,2,4 Matthieu Bueche and Mauro Tonolla^1,2,3

1Laboratory of Applied Microbiology (LMA), Department for environment constructions and design (DACD), University of applied sciences and arts of southern Switzerland (SUPSI), via Mirasole 22a, 6500 Bellinzona, Switzerland

2Microbiology Unit, Department of Botany and Plant Biology, University of Geneva, 1211 Geneva, Switzerland

3Alpine Biology Center Foundation, via Mirasole 22a, 6500 Bellinzona, Switzerland

4Federal Office for Civil Protection, Spiez Laboratory, Biology Division, 3700 Spiez, Switzerland

Flow cytometry as a tool to investigate the anoxygenic phototrophic sulfur bacteria coexistence in the chemocline of meromictic Lake Cadagno

Thesis

Reference

Flow cytometry as a tool to investigate the anoxygenic phototrophic sulfur bacteria coexistence in the chemocline of meromictic Lake

Cadagno

Flow Cytometry as a Tool to Investigate the Anoxygenic Phototrophic Sulfur Bacteria Coexistence in the Chemocline of

Meromictic Lake Cadagno

ACKNOWLEDGMENTS

TABLE OF CONTENTS

RÉSUMÉ

SUMMARY

ABBREVIATIONS

LIST OF TABLES AND FIGURES

INTRODUCTION

CHAPTER 1

Phylogenetic diversity of the microbial community in the water column of meromictic Lake Cadagno and evidence for seasonal dynamics