Community composition and interactions of biofilm bacteria on submerged freshwater
macrophytes
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)
an der Universität Konstanz
Mathematische-Naturwissenschaftliche Sektion Fachbereich Biologie
vorgelegt von Melanie Hempel
Konstanz, April 2008
Tag der mündlichen Prüfung: 06. Oktober 2008 Referent: PD Dr. Elisabeth Groß
Referent: PD Dr. Hans-Peter Grossart Referent: Prof. Dr. B. Schink
Success consists of going from failure to failure without loss of enthusiasm.
Winston Churchill (1874-1965)
Abbreviations i Summary in German – deutsche Zusammenfassung iii Summary in English – englische Zusammenfassung vii
I General Introduction 1
II Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater
Abstract 21
Introduction 22
Material & Methods 25
Results 28
Discussion 34
III Bacterial community composition of biofilms on two submerged macrophytes and an artificial surface in Lake Constance
Abstract 43
Introduction 44
Material & Methods 47
Results 51
Discussion 60
Supplementary 64
IV Spatio-temporal dynamics of the bacterial biofilm on two freshwater macrophytes and an artificial substrate in Lower Lake Constance
Abstract 69
Introduction 70
Material & Methods 72
Results 75
Discussion 85
Abstract 95
Introduction 96
Material & Methods 98
Results 100
Discussion 107
VI Single- and multispecies biofilm formation of tannin degrading bacteria on an aquatic macrophyte
Abstract 115
Introduction 116
Material & Methods 118
Results 121
Discussion 125
VII General Discussion and Perspectives 129
VIII References 139
Record of Achievement 159
List of Publications 161
Acknowledgement – Danksagung 165
Curriculum vitae 167
AFDM Ash free dry mass ANOVA Analysis of variance
BCC Bacterial community composition BLAST Basic local alignment search tool
CFB Cytophaga-Flavobacteria-Bacteroidetes CLSM Confocal laser scanning microscopy
DAPI 4ʹ,6–Diamidino–2–phenylindol
DGGE Denaturing gradient gel electrophoresis
dm Dry mass
DNA Deoxyribonucleic acid DOC Dissolved organic carbon DOM Dissolved organic matter
EPS Exopolysaccharide
FA Formamide
FISH Fluorescence in situ hybridization GIS Geographic information system HPLC High performance liquid chromatography
IGKB Internationale Gewässerschutzkommission für den Bodensee MICRO-FISH Microautoradiography combined with FISH
NaPPi Sodium pyrophosphate
NMDS Non metric dimensional scaling
OD600nm Optical density at 600 nm
PCR Polymerase chain reaction PVPP Polyvinylpolypyrrolidon
RFLP Restriction fragment length polymorphism rRNA Ribosomal ribonucleic acid
SB Schaproder Bodden
SD Standard deviation
SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis SEM Scanning electron microscope
TEP Transparent exopolymer particles
UPGMA Unweighted pair group method with arithmetic mean
Ziel dieser Arbeit war es, die bakterielle Biofilmgemeinschaft auf aquatischen Oberflächen, insbesondere von submersen Makrophyten, zu untersuchen. Im Mittelpunkt standen dabei die Zusammensetzung und Sukzession des bakteriellen Biofilms, der mögliche Einfluss von Umweltfaktoren, Habitat und der Pflanzen (Substrat) auf den Biofilm und die Interaktionen einzelner Isolate untereinander und mit aquatischen Herbivoren. In der Litoralzone von Seen bieten Makrophyten eine große Oberfläche zur Besiedlung von Bakterien und Algen. Zwischen Pflanzen und Epiphyten finden häufig Interaktionen statt, die für beide Seiten positiv oder auch negativ sein können. Interaktionen zwischen Pflanzen und epiphytischen Biofilm können z.B. durch Oberflächenveränderungen oder ausgeschiedene organische Substanzen stattfinden. Besonders Sekundärstoffe von Pflanzen (z.B. Phenole) sind bekannt dafür, dass sie andere Phototrophe oder Mikroorgansimen beeinflussen können.
Ich erwartete, dass das phenolreiche Tausendblatt Myriophyllum spicatum L. eine andere Biofilmzusammensetzung hat als das Laichkraut Potamogeton perfoliatus, die Armleuchteralge Chara aspera oder künstliche Substrate (Plastikstreifen).
Myriophyllum spicatum scheidet algizide und bakterizide Polyphenole aus, während einige Chara-Arten algizid wirkende zyklische Thiane produzieren. Für Potamogeton perfoliatus ist nicht bekannt, dass es Polyphenole synthetisiert und Bakterien oder Algen im Wachstum hemmt. Da M. spicatum zudem einen deutlichen Gradient von Makronährstoffen und Polyphenolen von den jungen Apikalmeristemen zu den älteren Blättern hinweg aufweist, war ein weiterer Aspekt dieser Arbeit, den Einfluss des Blattalters der Pflanzen auf die Biofilmzusammensetzung zu untersuchen. Chara aspera und M. spicatum kommen sowohl im Bodensee (Süßwasser) als auch im Schaproder Bodden (Brackwasser) vor. Daher haben wir die bakterielle Biofilmzusammensetzung auf beiden Pflanzen in beiden Habitaten verglichen. Alle Biofilmanalysen dieser Arbeit sind mit molekularen Methoden wie FISH
(Denaturierende Gradienten Gel Elektrophorese) und der Erstellung einer Klonbibliothek durchgeführt worden. Durch die Kombination mehrerer Methoden wurden die erhaltenen Ergebnisse bestätigt und eventuelle Schwachstellen einer Methode ausgeglichen.
Alle Untersuchungen des bakteriellen Biofilms lassen darauf schließen, dass die Biofilme auf den jeweiligen Substraten von Bakterien der CFB-Gruppe dominiert waren. Alpha- und Betaproteobakterien waren am zweithäufigsten, während Planktomyceten fast nur auf C. aspera im Schaproder Bodden gefunden wurden. Wir konnten einen deutlichen Einfluss des Habitats und des Substrates auf Planktomyceten nachweisen, während CFB-Bakterien eher durch die Pflanze und das Blattalter beeinflusst wurden.
Beim Vergleich des Biofilms auf M. spicatum mit dem auf P. perfoliatus und Plastikstreifen wurde deutlich, dass die Jahreszeit keinen Einfluss auf die Zusammensetzung des bakteriellen Biofilms hat. Umweltfaktoren wie Wasserstand und –temperatur, Leitfähigkeit, pH und der Kohlenstoff- und Gesamtpolyphenolgehalt der Pflanze haben hingegen einen Einfluss. Die bakterielle Biofilmgemeinschaft auf P. perfoliatus und den Plastikstreifen ähnelte einander mehr als diejenige auf M. spicatum. Alle Ergebnisse aus den Analysen der Biofilmgemeinschaften deuten darauf hin, dass gerade Apikalmeristeme von M.
spicatum einen sehr heterogenen und speziellen Biofilm haben. Wir vermuten, dass dies mit dem erhöhten Polyphenolgehalt in diesen Abschnitten zusammenhängt. Die Ergebnisse der Klonbibliothek bestätigen dies, und laut den Sequenzvergleichen mit GenBank liegen viele dieser Biofilmbakterien auch größtenteils noch nicht in Kultur vor.
Wir konnten drei Isolate aus dem Biofilm (Pantoea agglomerans & Agrobacterium vitis) und dem Umgebungswasser (Matsuebacter sp.) von M. spicatum isolieren, die
wir untersucht, ob diese Isolate axenische M. spicatum besiedeln können.
Da Epiphyten zwangsläufig von Herbivoren aufgenommen werden, können diese so deren Verdauung und Darmflora beeinflussen. In Fütterungsexperimenten testete ich, ob das polyphenolabbauende Bakterium Matsuebacter sp. einen Einfluss auf das Larvenwachstum des Wasserzünslers Acentria ephemerella (DENIS &SCHIFFERMÜLLER) hat.
Das Wachstum der Larven wurde im Vergleich zu axenischem M. spicatum weder negativ noch positiv beeinflusst, wenn sie mit Matsuebacter sp. besiedeltem M.
spicatum gefüttert wurden. Daraus schließen wir, dass Matsuebacter sp. weder als zusätzliche Nährstoffquelle dient, noch die vorhandene Darmflora der Larve beeinflusst oder nennenswert die Polyphenole verändert hat.
Während der Einfluss von Matsuebacter sp. auf das Larvenwachstum vernachlässigbar war, ist es uns gelungen, eine dichte und schnelle Biofilmbildung dieses Bakteriums auf axenischem M. spicatum zu zeigen. Dadurch wird die Biofilmbildung des landwirtschaftlich genutzten „Biocontrollers“ P. agglomerans gehemmt und diejenige des Pflanzenpathogens A. vitis verstärkt.
Mit dieser Arbeit ist es mir gelungen, einen Beitrag zu dem bisher recht spärlichen Wissen über bakterielle Biofilm auf aquatischen Pflanzen zu leisten, weiterhin die Interaktionen einzelner Bakterien miteinander zu beleuchten und auch den Bogen zu höheren trophischen Ebenen zu spannen. Einzelne Bakteriengruppen werden offensichtlich von dem jeweiligen Substrat und Habitat beeinflusst, während für ganze Bakteriengemeinschaften auch Umweltparameter wie Wasserstand und–
temperatur und Leitfähigkeit, aber auch der Kohlenstoffgehalt und Gesamtphenolgehalt der Pflanzen von Bedeutung sind.
The aim of my PhD thesis was to investigate the bacterial biofilm community composition (BCC), especially on submerged macrophytes. The special interest was the composition and succession of the heterotrophic biofilm and possible influences such as environmental factors, habitat and plants (substrate) on the biofilm and the interaction of isolates with each other and with aquatic herbivores. On the littoral zones of lakes, macrophytes offer a large area for colonization of bacteria and algae.
Interactions between plant and epiphytes are frequent and can be positive and negative for both sides. Interactions between macrophytes and epiphytic biofilm can be mediated by structural changes of the surface or by exuded organic compounds.
Especially secondary metabolites of Plants (e.g., phenols) are known to have an impact on other phototrophs or microorganisms.
I expected that the phenol-rich milfoil Myriophyllum spicatum L. would have a different BCC than the pondweed Potamogeton perfoliatus, the stonewort Chara aspera or artificial substrates (polypropylene sheets). Myriophyllum spicatum exudes algicidal and bactericidal polyphenols, while some Chara species produce algicidal cyclic sulphur compounds. It is not known if P. perfoliatus synthesizes polyphenols and if it may inhibit bacterial and algal growth. Another aspect of this work was to investigate the influence of leaf age on the BCC, since M. spicatum displays a distinct gradient of macronutrients and polyphenols from young apical meristems to older leaves. Both Char aspera and M. spicatum occur in Lake Constance (freshwater) and in the Schaproder Bodden (brackish water). Thus, we compared the BCC on both macrophytes in both habitats. All analyses of the BCC in this study have been done with FISH (fluorescence in situ hybridization) and in Lake Constance additionally with DGGE (denaturing gradient gel electrophoresis) and the construction of a clone library. With the combination of several methods, I could verify the obtained results and possible disadvantages of one method could be evened out by the other.
the respective substrates were dominated by bacteria of the CFB-group. Alpha- and betaproteobacteria were the second most abundant groups, while planctomycetes were only found on brackish water C. aspera. Planctomycetes were largely influenced by the habitat and the substrate type (plant species) while bacteria of the CFB-group were rather influenced through plant species and leaf age.
The BCC comparison on M. spicatum, P. perfoliatus and the artificial substrates was not influenced by season. However, environmental factors such as water level and temperature, conductivity, pH and the carbon and total phenolic content of the plant tissue influenced the bacterial biofilm community composition. The BCC on the artificial substrates was more similar to that on P. perfoliatus than to that on M spicatum. The results obtained in all those community studies revealed a rather distinct and heterogeneous BCC on M. spicatum apices. We assume that this is a consequence of the high polyphenol content in these plant parts. The data obtained in the clone library support this finding. According to GenBank, most of the sequences obtained in the clone library do belong to bacteria not yet cultured.
We were able to isolate three bacterial strains from the biofilm (Pantoea agglomerans
& Agrobacterium vitis) and the surrounding water (Matsuebacter sp.) of M. spicatum.
All three are able to degrade polyphenols. With an especially designed experimental set-up, we tested if the three isolates were capable to colonize axenic M. spicatum.
Since epiphytes are taken up inevitably during feeding of herbivores they may have an impact on digestion and gut microbiota. In no choice feeding experiments I investigated, if the polyphenol degrading Matsuebacter sp. has an impact on the larval growth of the aquatic moth Acentria ephemerella (DENIS &SCHIFFERMÜLLER).
In comparison to axenic M. spicatum, plants colonized with Matsuebacter sp. had no negative or positive impact on larval growth. Thus we conclude that Matsuebacter sp.
neither serves as an additional nutrient source, nor influences the gut microbiota or alters the exuded plant polyphenols.
prove that this bacterium forms dense biofilms on M. spicatum rather quickly. The presence of Matsuebacter sp. reduces the biofilm formation of the agriculturally used bio control agent P. agglomerans, and thus that of the plant pathogen A. vitis is enhanced.
With this work, I contributed to the scarce knowledge on bacterial biofilms on aquatic plants. Further I elucidated the biofilm formation and interactions of single strains and investigated their impact on higher tropic levels. Single bacterial groups are obviously influenced by the substrate and habitat type. Bacterial communities in their whole are rather determined by environmental factors like water level and temperature, and conductivity, and also the plant carbon and total phenolic content.
Chapter I
General Introduction
FUNCTIONS OF MACROPHYTES IN LITTORAL ZONES OF LAKES
In the littoral zones of lakes, macrophytes are the major primary producers and a vital structural element. Especially in shallow eutrophic lakes, they often are responsible for clear water states, caused by light and nutrient competition advantages over algae or allelopathy (Scheffer et al. 1993, Hilt & Gross 2008).
Submerged macrophytes reduce sediment resuspension. They transport selected mineral nutrients taken up by the root through the lacunar system to the leaf surface. For example, methane and manganese are released in substantial amounts for epiphytic bacteria and algae (Jackson et al. 1994, Schuette 1996, Heilman &
Carlton 2001). Besides providing shelter for young fish and zooplankton, macrophytes are also frequently consumed by waterfowl. Macrophytes provide a large surface area for egg deposition of snails and fish and for the colonization of microorganisms such as algae, fungi and bacteria. Thus, they fulfil many functions in this heterogeneous and active habitat.
With a total volume of 48.4 km³, the prealpine Lake Constance contributes 0.04% of all freshwater lakes on earth (Figure 1.1A). Its shore line is 273 km long and comprises the larger and deeper oligotrophic Upper Lake (186 km shoreline;
101 mean depth; 8 mg total phosphorus m-3) and the smaller mesotrophic Lower Lake (87 km shoreline; 13 m mean depth; 17 mg total phosphorus m-3; (IGKB 2006, 2007)). Especially in the Upper Lake, the phosphorus concentration has been declining constantly in the past years.
In lakes, the littoral zone is usually defined as the near shore area where sunlight penetrates all the way to the sediment and allows macrophytes or other benthic primary producers to grow. Light levels of about 1% or less of surface values usually define this depth. In 1993, the littoral zone of Lower Lake was 18 km² as determined by GIS measurements (geographic information system;
(Schmieder 1997)).
According to this method, the Upper Lake had littoral zone of 57 km² contrasting 18 km² in the Lower Lake with the littoral zone defined as the area above 10 m water depth. Due to the ongoing reoligotrophication, light is no longer a limiting factor for macrophyte growth and with an increased maximum depth of macrophytes, the littoral zone of Lower Lake has most likely increased.
The sampling site of this study was located in lower Lake Constance near a gravel ridge close to the Island of Reichenau in 2 – 3 m water depth (N47°42,247, E9°02,289; Figure 1.1Band C). This site is at the wind exposed north-western part of the island and water currents are higher than in the northern part. While Myriophyllum spicatum (Figure 1.2A) is found on the ridge or on the slope of it, alongside the ridge dense mats of Chara spp. cover the sandy sediment (Figure 1.2B). In this bay two larger Potamogeton perfoliatus stands (Figure 1.2C) are located close to the shore on the right and left hand side of the ridge.
Figure 1.1. Lake Constance and study site A) Lake Constance (Circle indicates Island of Reichenau) B) Island of Reichenau C) Study site located at Niederzell.
Pictures are taken from: A) www.igkb.de B) ww.insel-reichenau.de
C) Dr. T. Heege, DLR Oberpfaffenhofen.
Figure 1.2. Aquatic substrates investigated in this study. A) Myriophyllum spicatum stand in a dense Chara sp. mat B) Chara sp. C) Potamogeton perfoliatus patch C) Exposed polypropylene sheets used as artificial substrates. All pictures by the courtesy of Dr. M. Mörtl.
IMPORTANCE OF BACTERIAL BIOFILMS IN AQUATIC HABITATS
Bacteria occur ubiquitous in nature. They are found in soil, marine and freshwater, sewage sludge and even in extreme environments such as hydrothermal vents (Hugenholtz et al. 1998). In the last decades, it has been commonly acknowledged that bacteria prefer an attached life style if nutrient conditions are favourable and thus are mainly found on surfaces (Costerton et al.
1995, Stanley & Lazazzera 2004). Since then, much research has been done on biofilms, their role and function in wastewater treatment, health care, industries and ecology (Paerl & Pinckney 1996, Morris & Monier 2003, Pasmore & Costerton 2003, Parsek & Fuqua 2004, Stanley & Lazazzera 2004). Bacteria, if in biofilms or not, contribute to the overall nutrient cycling in pelagic and littoral zones, and in streams (Ardon & Pringle 2007). They are involved in the degradation of sinking
metabolites that can be used further and are the essential part of the microbial loop (Riemann et al. 2000, Cotner & Biddanda 2002). In industrial systems and in water supply biofilms often cause problems and immense costs since they are hard to remove, induce corrosion and are a source of contamination (Pasmore &
Costerton 2003).
The formation of biofilms has many ecological implications for bacteria and their surfaces. Biofilms found in nature are usually multispecies biofilms, embedded in a matrix of extracellular polymeric substances (EPS), in which bacteria with different metabolically characteristics coexist and may act as symbionts (Eberl 1999, Burmolle et al. 2006). The EPS matrix surrounding the biofilm prevents the cells from desiccation, and channels that form in the matrix are used to transport substrates or proteins, which would otherwise be lost in the surrounding water (Czaczyk & Myszka 2007). It is well known from clinical studies that bacterial biofilms are more resistant to antibiotics or detergents than single cells due to the surrounding matrix (Stewart & Costerton 2001, Burmolle et al. 2006, Harrison et al. 2007). This aspect can certainly be transferred to nature since bacteria are often exposed to bactericidal substances in the environment (Burmolle et al. 2006). Bacteria in biofilms are also more resistant to grazing by flagellates. Assumingly, this is caused by the thickness of the biofilm and the EPS–
matrix, which makes them less accessible (Jürgens & Matz 2002). Further, biofilms provide a very structured and heterogeneous habitat on a very small scale. Cells at the bottom of the biofilm often experience anaerobic conditions while cells in the outer layers are more exposed to grazing, toxins or UV–radiation.
One mechanism in biofilm formation that has received much attention in the past years is quorum sensing. It is defined as cell density dependent gene regulation and is mediated via signal molecules, e.g., N–acylhomoserinlactones in gram–
negative or peptides or butyrolactone in gram–positive bacteria (Bibb 1996, Kleerebezem et al. 1997, Chhabra et al. 2005). Several functions have been shown to be regulated by quorum sensing, among them siderophore production, motility,
pathogenicity, bioluminescence, biofilm formation and EPS production (Guan et al. 2000, Hammer & Bassler 2003, Lupp et al. 2003, Marketon et al. 2003). In the past, quorum sensing–studies focussed mainly on medical implications. Studies on the ecological relevance of quorum sensing are scarce (Manefield & Turner 2002) but gave hints that it might not be as important as assumed (Styp von Rekowski et al. 2008). In preliminary experiments, I tested whether bacteria capable to use tannic acid as a sole carbon source produce N–acylhomoserinlactones. In bioassays, I found several strains that produce those signal molecules but found no evidence that those strains grew faster than others that did not produce these compounds. Other studies found that plant derived furanones and polyphenols may interfere with quorum sensing regulated biofilm formation (see below;
(Hentzer et al. 2002, Huber et al. 2003)). Thus, plants and algae have developed strategies against this mechanism, indicating that the ecological role of quorum sensing is controversial and needs further investigation.
Studies on biofilms (initial colonization, succession, interactions) are often conducted in artificial systems with only two or three strains. The advantage of those set ups is that all environmental parameters can be kept constant. Further, the selected strains can be modified genetically, which is attractive to investigate the importance of certain genes in biofilm formation or interactions between different bacteria in the biofilm. Biofilms are usually investigated by a combination of different dyes and microscopy. With the introduction of fluorescence in situ hybridization, green fluorescent protein labelled strains and confocal laser microscopy it has become possible to observe biofilm formation from the beginning on in situ. Researchers have shown that biofilms do not consist of several flat layers but form mushroom like structures that are connected with transport channels (De Beer et al. 1994, Picioreanu et al. 2000). Further, biofilms are no static communities but are shaped by settling and sloughing of organisms.
Thus, biofilms are characterized by a constantly changing community composition in which participants have to react and act constantly to new metabolic partners or
even competitors from the beginning on (Grossart et al. 2003, Kiorboe et al. 2003, Pasmore & Costerton 2003).
On the surface of M. spicatum, bacteria will also live as a biofilm ‘entity’ that interacts in multiple ways To investigate biofilm formation of selected strains and their interactions on M. spicatum leaves, we developed a set up, which allows to do exactly this. We chose three strains originating from the surrounding water (Matsuebacter sp.) and the biofilm of M. spicatum (Agrobacterium vitis and Pantoea agglomerans) all with different characteristics and abilities to degrade polyphenols.
Matsuebacter sp. (Mitsuaria sp.; betaproteobacteria) is able to constitutively degrade polyphenols (Müller et al. 2007), while A. vitis, a grapevine pathogen, can only degrade polyphenols if the degradation pathways are induced (Ophel & Kerr 1990, Müller et al. 2007). The gammaproteobacterium P. agglomerans is frequently used in agriculture as a biological control agent and possesses the required enzymes for phenol degradation (Zeida et al. 1998). It is further found throughout the world in the gut of ruminants and herbivores (Nelson et al. 1998, Pidiyar et al.
2004).
INTERACTIONS OF BACTERIA WITH PHOTOTROPHS AND HERBIVORES In aquatic systems it is well known that epiphytes and plants do interact in various ways (Beattie & Lindow 1999, Kubanek et al. 2003, Mathesius et al. 2003).
In general it can be assumed that epiphytes have quite an impact on host organisms and vice versa.
As mostly sessile organisms, plants have to defend themselves against potential enemies, e.g., herbivores, epiphytic algae and pathogenic bacteria or fungi. Thus, they developed several methods to hold off enemies. Most prominent examples are thorns or spikes, thick leaves and also chemical defence. In aquatic systems, chemical defence is most commonly used against other phototrophs or microorganisms. Chemical defence results in compounds that taste bad to herbivores, or inhibit epiphytic phototrophs or heterotrophic microorganisms. In 1937, Hans Molisch coined the term allelopathy for plant-plant and plant-bacteria interactions. Allelopathy covers biochemical interactions, both stimulatory and inhibitory, among different primary producers or between primary producers and microorganisms (Molisch 1937). Here, we focus on and elucidate the impact of allelochemicals produced by different aquatic plants on the bacterial community composition in the epiphytic biofilm, especially those of Myriophyllum spicatum.
Since organic metabolites always leak from the macrophyte tissue (Godmaire &
Planas 1986), epiphytic bacteria should be directly affected by these compounds.
Biofilms on plants may have beneficial or detrimental effects to their host. First of all, they reduce the light availability of the plant, especially if algae or phototrophic bacteria are embedded. Further, the biofilm may contain pathogens that invade plant cells or promote biofouling. On planktonic algae, attached bacteria increase the sinking speed, and the algae reach light limitation earlier than without the biofilm (Grossart et al. 2005). On the other hand, the biofilm may lead to reduced grazing, and the plant can benefit from degraded compounds and CO2
produced by bacteria. Phototrophs might even influence the bacterioplankton community composition. The macrophyte Vallisneria americana influences the
bacterioplankton community indirectly over DOC (dissolved organic carbon) and phosphorus release (Huss & Wehr 2004). If the ammonia level was high, the bacterioplankton numbers were positively influenced by DOC and phosphorus release of the plant. If ammonia was limiting, non–rooted plants had a negative effect on bacterioplankton abundance, while rooted macrophytes had no effect.
Further, the mode of interaction between plants and phototrophs can be influenced by the trophic state (Danger et al. 2007a, Danger et al. 2007b). Here, nitrogen limitation of algae resulted in commensalism, phosphorus limitation in competition and nutrient rich situations in mutualism.
In the past, many studies described allelochemical interactions between bacteria and phototrophic organisms. The marine red algae Bonnemaisonia hamifera and Delisea pulchra produce and release furanones that inhibit the settling of bacteria by interfering with quorum sensing regulated biofilm formation (Maximilien et al.
1998, Huber et al. 2003, Nylund et al. 2005). These furanones also affect the swarming motility of Serratia liquefaciens and indirectly larval attachment (Rasmussen et al. 2000, Dobretsov et al. 2007). Positive effects of bacteria were found for Roseobacter gallaciencis and Pseudoalteromonas tunicata on the seaweed Ulva australis where they produce antifouling compounds (Rao et al. 2006). Axenic Ulva linza need bacteria to promote growth and restore their growth form (Matsuo et al. 2005, Marshall et al. 2006).
For all plant species investigated in this study, the existence of allelopathic compounds has been reported. In Eurasian watermilfoil Myriophyllum spicatum, tellimagrandin II, a hydrolysable polyphenol (Figure 1.3A), is present in concentration of 5% of the dry mass (up to 25% of all tannins on apices and leaves), and is responsible for inhibition of the photosystem II in cyanobacteria (Leu et al. 2002). Further, plant polyphenols inhibit the gut microbiota of the herbivorous larvae Acentria ephemerella (DENIS &SCHIFFERMÜLLER, Figure 1.4A and B), and are supposed to retard the growth of this larvae (Choi et al. 2002, Walenciak et al. 2002). To compare the impact of plant chemistry on the bacterial
community composition of M. spicatum, we chose the pondweed Potamogeton perfoliatus that contains hardly any phenolic compounds (Choi et al. 2002). It grows in the vicinity of M. spicatum and although algae-inhibiting compounds, mostly di-terpenes, have been described for several Potamogeton species, none of these effects or compounds have been described and isolated from P. perfoliatus (DellaGreca et al. 2001). We also investigated the bacterial community composition of a third macrophyte, the macroalgae Chara aspera. Some Chara species contain cyclic sulphur compounds that inhibit bacteria and algae (Figure 1.3C; (Anthoni et al. 1987)).
The herbivore Acentria ephemerella causes substantial damage to aquatic plants such as P. perfoliatus and M. spicatum (Gross et al. 2002). Although it fully develops on the latter, its growth is retarded compared to polyphenol-free P. perfoliatus
Figure 1.3. Chemical compounds in freshwater macrophytes.
A) Tellimagrandin II B) Tannic acid (http://www.pharmainfo.net) C) Trithiane and dithiane from Chara globularis.
S S S S
C H3
C H3 S
S S A)
B)
C)
assumingly due to the high polyphenol content of M. spicatum (Choi et al. 2002).
Many studies showed the importance of gut bacteria for insects, esp. herbivores (Ji et al. 2000, Ji & Brune 2001, Dillon & Dillon 2004) and it has been assumed that the negative impacts of plant tannins on herbivores are often a consequence of plant- bacteria interactions (Schultz et al. 1992). This could especially be the case for bacteriostatic hydrolysable polyphenols. In the case of A. ephemerella, isolated gut bacteria were inhibited in the presence of M. spicatum-derived tannins. To test if tannin-degrading bacteria will have an influence on the growth of A. ephemerella larvae, we conducted no choice experiments in which larvae were fed Matsuebacter sp.-colonized M. spicatum leaves in comparison to axenic plants or those colonized by a natural bacterial biofilm. Matsuebacter sp. grows constitutively with tannic and gallic acid (see above).
Figure 1.4. A) Aquatic moth Acentria ephemerella B) A. ephemerella larvae of different instar ages (courtesy of Dr. E.M. Gross)
BACTERIAL GROUPS ON DIFFERENT SURFACES AND HABITATS
Surprisingly, if the ISI web of science database is searched for the terms
‘bacteria’, ‘biofilm’, ‘freshwater’, ‘community’ and ‘composition’ in the years 1956 to 2008 (week 16), only nine hits are displayed. If the term ‘freshwater’ is not included, the number of hits rises to 116. This is surprising, since biofilms have received much attention in the last decade but obviously, freshwater biofilms did not. Many studies that deal with freshwater biofilms often focus on certain bacterial groups rather than on whole communities (Brümmer et al. 2004,
Tadonléké 2007). Thus, studies describing whole biofilm communities are scarce, especially on macrophytes.
The invention and introduction of molecular methods to microbial ecology has increased our knowledge of bacterial communities immensely over the last two decades (Head et al. 1998). Especially methods like denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridisation (FISH) contributed to this gain in knowledge. They allow to process large sample numbers and provide data not only of community structures (DGGE), but also on species composition and spatial distribution within a given community (FISH).
In the past, many aquatic bacterial communities have been described on the basis of these new methods in various habitats, giving evidence that most aquatic bacteria occur throughout the world, though in various abundances.
Betaproteobacteria, for example, are commonly found in freshwater habitats, while they do not occur in the marine bacterioplankton (Glöckner et al. 1999).
Aquatic biofilm communities have been described on and in various substrates such as sediments, chlorophytes, diatoms, sponges, lake and marine snow, stones, steel foil, ceramic tiles or propylene sheets in marine and freshwater by various molecular methods for the 16S rRNA gene or functional genes.
On green algae and diatoms as well as on lake snow and copepod carcasses, researchers found high abundance of the different proteobacteria groups and of Cytophaga-Flavobacteria-Bacteroidetes (CFB). The abundance of different groups was somewhat dependent on the substrates investigated. Lake and marine snow are often composed of dead zooplankton or algae, but also transparent exopolymer particles (TEP) are important hot spots for the microbial degradation of organic matter (Simon et al. 2002). On Lake Constance lake snow, mainly alpha- and betaproteobacteria were found in the epilimnion, while in the hypolimnion bacteria of the CFB group were dominant (Schweitzer et al. 2001).
Gammaproteobacteria were of minor importance on particles from all depth investigated. The researchers assume that a limited number of alpha- and
betaproteobacteria first consume and then release amino acids, while in deeper water layers CFB-bacteria consume the refractory components. On diatom microaggregates, initially alphaproteobacteria dominate, while betaproteobacteria and CFB increase towards the end. Brachvogel and colleagues defined four types of microaggregates with different bacterial communities. Here, betaproteobacteria dominated particles originating from diatoms, while on “DAPI yellow particles”
betaproteobacteria and CFB-bacteria were equally abundant. Interestingly, gammaproteobacteria, which were not numerous in other studies, were most abundant on particles originating from zooplankton and phytoplankton (62% of DAPI counts; (Brachvogel et al. 2001)). Although in all these ‘Lake Constance’
studies only probes for alpha-, beta- and gammaproteobacteria and CFB were used, these groups accounted for more than 50% of all cells detected and thus, the community on lake snow aggregates in Lake Constance seems to be made out of a few bacterial groups.
While community composition of aggregated bacteria in Lake Constance seemed rather constant, the community composition of aggregated bacteria in Lake Baikal was different. Here, gammaproteobacteria were quite abundant in all water layers, except for 100 m depth. Bacteria of the CFB-group were not as abundant and betaproteobacteria increased with depth (Ahn & Burne 2007).
Several studies dealing with freshwater biofilms in streams found high abundances of betaproteobacteria and CFB. Alphaproteobacteria were frequently found while gammaproteobacteria were of minor importance (Brummer et al.
2000, Olapade & Leff 2004, 2005, 2006). Olapade and co-workers proved very nicely that the BCC depends on the type and quality of dissolved organic matter (DOM) provided. Labile DOM (e.g., glucose) evoked more response in all bacterial groups investigated than leaf leachate, algal exudates or inorganic nutrients (Olapade & Leff 2005).
Descriptions of the heterotrophic biofilm community composition (BCC) on macrophytes are scarce but do exist for Myriophyllum spicatum and some
Potamogeton species. The BCC on M. spicatum has been described by cultivation based methods at the beginning of 1990 (Chand et al. 1992) and again with DGGE in 2000 (Walenciak 2004). The latter study described differences between attached and free living communities.
AIMS OF THIS STUDY
The main objective of my Ph.D. thesis was to increase the scarce knowledge on bacterial biofilms on freshwater macrophytes and to investigate patterns and possible processes determining their community structure. Whole community compositions on different macrophytes in different habitats have not been described before. Although environmental factors as DOM, chlorophyll content and conductivity have been identified to determine differences of bacterial communities from different habitats, so far no one attempted to investigate the influence of plant age, plant species and plant chemistry on the bacterial community composition (BCC). It is important to understand internal and external influences on bacterial dynamics, since they might further influence higher trophic levels.
Based on my initial studies in Lake Constance, we asked how BCC is influenced by different habitats and plants (Chapter 2). We chose a higher plant (Myriophyllum spicatum) and a macroalga (Chara aspera), both occurring in the same habitats (Lake Constance and Schaproder Bodden). In cooperation with Maja Blume from the University of Greifswald, we investigated differences and similarities of the BCC on young and old plant parts of both macrophytes in both habitats with fluorescence in situ hybridization (FISH).
In Chapters 3 and 4, I focus on spatial and temporal differences of the BCC on M. spicatum in comparison to other substrates. First, I investigated the seasonal and within-plant differences of the BCC on this macrophyte with denaturing gradient gel electrophoresis (DGGE) during summer 2005. I extended these
substrate, the plant age (apices and leaves) or the season. Environmental parameters and plant chemistry were measured as potential determining factors. I investigated the BCC on Myriophyllum spicatum, Potamogeton perfoliatus and polypropylene sheets used as artificial substrate (Figure 1.2D) with DGGE (Chapter 3) and FISH (Chapter 4).
Herbivorous insects may take up biofilm bacteria with their host plants. We knew that M. spicatum derived tannins decrease the growth of larvae of the aquatic moth Acentria ephemerella and inhibit selected strains of the gut microbiota. We further had bacteria isolated from M. spicatum stands that are able to degrade the plants’ tannins. Therefore, I compared the impact of axenic (= bacteria-free) plants to those with a biofilm of Matsuebacter sp. or plants with a natural bacterial biofilm on the growth of A. ephemerella larvae in no choice feeding experiments (Chapter 5).
Finally, we were interested in the recolonization of M. spicatum with previously isolated bacterial strains and how these strains would interact (Chapter 6). In her diploma thesis, Sonja Wicks developed, based on my findings and with my guidance, an experimental set up to follow the recolonization of M. spicatum and the interactions of the different proteobacterial strains Matsuebacter sp., Agrobacterium vitis and Pantoea agglomerans by DAPI staining and FISH.
Chapter II
Epiphytic bacterial community composition on two common submerged macrophytes in brackish water and freshwater
Melanie Hempel, Maja Blume, Irmgard Blindow & Elisabeth M. Gross BMC Microbiology 8(1):58
ABSTRACT: Plants and their heterotrophic bacterial biofilm communities possibly strongly interact, especially in aquatic systems. We aimed to ascertain whether different macrophytes or their habitats determine bacterial community composition. We compared the composition of epiphytic bacteria on two common aquatic macrophytes, the macroalga Chara aspera Willd. and the angiosperm Myriophyllum spicatum L., in two habitats, freshwater (Lake Constance) and brackish water (Schaproder Bodden), using fluorescence in situ hybridization. The bacterial community composition was analysed based on habitat, plant species, and plant part.
The bacterial abundance was higher on plants from brackish water [5.3×107 cells (g dry mass)–1] than on plants from freshwater [1.3×107 cells (g dry mass)-1], with older shoots having a higher abundance. The organic content of freshwater plants was lower than that of brackish water plants (35 vs. 58%), and lower in C. aspera than in M. spicatum (41 vs. 52%). The content of nutrients, chlorophyll, total phenolic compounds, and anthocyanin differed in the plants and habitats. Especially the content of total phenolic compounds and anthocyanin was higher in M. spicatum, and in general higher in the freshwater than in the brackish water habitat. Members of the Cytophaga–
Flavobacteria–Bacteroidetes group were abundant in all samples (5–35% of the total cell counts) and were especially dominant in M. spicatum samples.
Alphaproteobacteria were the second major group (3–17% of the total cell counts). Betaproteobacteria, gammaproteobacteria, and actinomycetes were present in all samples (5 or 10% of the total cell counts). Planctomycetes were almost absent on M. spicatum in freshwater, but present on C. aspera in freshwater and on both plants in brackish water.
Bacterial biofilm communities on the surface of aquatic plants might be influenced by the host plant and environmental factors. Distinct plant species, plant part and habitat specific differences in total cell counts and two bacterial groups (CFB, planctomycetes) support the combined impact of substrate (plant) and habitat on epiphytic bacterial community composition. The presence of polyphenols might explain the distinct bacterial community on freshwater M. spicatum compared to that of M. spicatum in brackish water and of C. aspera in both habitats.
INTRODUCTION
In aquatic systems, bacteria occur often associated with surfaces, e.g., in biofilms or on lake or marine snow (Costerton et al. 1995). Biofilm associated bacteria are most abundant at intermediate nutrient availability while either low or high nutrient conditions favour planktonic growth of bacteria (Stanley
& Lazazzera 2004). Biofilms are not only formed on abiotic surfaces but also on living organisms such as aquatic plants and algae.
In freshwater and marine habitats, bacteria associated with cyanobacterial blooms, diatom blooms, phytoplankton (Jasti et al. 2005), lake snow (Weiss et al. 1996), and bacterioplankton (Glöckner et al. 1999, Shade et al. 2007) have been investigated. Betaproteobacteria occur almost exclusively in freshwater but not in saline habitats, while alphaproteobacteria are more abundant in marine than in freshwater samples (Glöckner et al. 1999). Alphaproteobacteria dominate the planktonic bacteria in the North Sea, followed by the Cytophaga–Flavobacteria–Bacteroidetes (CFB) group, and all groups of bacteria display a seasonal succession (Sapp et al. 2007). Diverse bacterial communities dominate in cyanobacterial blooms, including members of the CFB group and betaproteobacteria (Eiler & Bertilsson 2004). Mainly members of the CFB group and alphaproteobacteria, especially Roseobacter, are attached to marine diatoms (Riemann et al. 2000, Grossart et al. 2005). Members of the CFB group and alpha-, beta-, and gammaproteobacteria have been identified by molecular methods on the chlorophytes Desmidium devillii, Hyalothexca dissliens, and Spondylosium pulchrum (Fisher et al. 1998). In general, the bacteria associated with diatoms and some chlorophytes that have been studied are mostly heterotrophic. In contrast, information about bacterial biofilms on aquatic macrophytes is scarce. A general overview and comparisons of attached and planktonic bacterial communities in freshwater and marine
habitats is given in (Simon et al. 2002, Pernthaler & Amann 2005) and references therein.
Submerged macrophytes are, in addition to algae, the main primary producers in lakes; they structure the littoral zone and prevent resuspension of sediments, thus enabling clear water states (Scheffer et al. 1993). The freshwater macrophytes Myriophyllum spicatum and Chara globularis, and possibly also other Chara species, produce secondary compounds such as polyphenols and cyclic sulfur compounds, which exert allelopathic activity against algae and cyanobacteria (Anthoni et al. 1987, Nakai et al. 2000).
Antibacterial cyclic quaternary amines have been isolated from C. globularis (Anthoni et al. 1987). Hydrolysable polyphenols of M. spicatum, especially tellimagrandin II, inhibit photosystem II of cyanobacteria (Leu et al. 2002).
Plant polyphenols may have antimicrobial activity, but some bacteria may also overcome polyphenol-based plant defences (Scalbert 1991).
Not only secondary metabolites but also nutrients possibly affect biofilm density and composition. Depending on their life cycle stage, macrophytes may release low to substantial amounts of macronutrients (Carignan & Kalff 1982), and at times high concentrations of micronutrients (Jackson et al. 1994).
Especially older plant parts may leak both organic compounds and inorganic nutrients (Sondergaard 1981). Nutrient conditions affect the impact of submerged macrophytes on bacterioplankton: Vallisneria americana has a positive impact on bacterioplankton density under high NH4+ conditions, but a neutral or negative impact when NH4+ is limiting (Huss & Wehr 2004).
Biofilms can be both beneficial and detrimental for submerged macrophytes.
On the positive side, epiphytic biofilms provide organic compounds and carbon dioxide to the macrophytes and enhance nutrient recycling (Wetzel 1993). Further, the biofilm bacteria Roseobacter gallaciencis and Pseudoalteromonas tunicata that colonize the marine alga Ulva australis produce
require bacteria to restore the typical growth form, and some bacteria even enhance the algal growth rate (Matsuo et al. 2005, Marshall et al. 2006).
Negative impacts on submerged macrophytes could arise from increased shading by thick biofilms and possibly also from pathogenic bacteria present in the biofilm. Macroalgae can also have negative effects on epiphytic bacteria.
For instance, bacterial colonization of the marine red algae Bonnemaisonia hamifera and Delisea pulchra is inhibited by algal-released secondary metabolites (Maximilien et al. 1998, Huber et al. 2003). These furanones also affect the swarming motility of Serratia liquefaciens (Rasmussen et al. 2000) and indirectly affect larval attachment (Dobretsov et al. 2007). Whether or not such chemical interactions between plants and bacteria are important for biofilm density and community composition on aquatic macrophytes is unknown. The only study addressing microbial diversity on M. spicatum showed that the biofilm was dominated by gammaproteobacteria and members of the CFB group (Chand et al. 1992). Bacterial epiphytes of C. aspera have not been described before.
Given that a strong interaction might exist between plants and their associated heterotrophic biofilm, especially in aquatic systems, we questioned whether different macrophytes (substrate, plant age) or the respective habitat determines bacterial community composition. We selected two common, allelochemically active, submerged macrophytes, Chara aspera and Myriophyllum spicatum, sampled in freshwater (Lake Constance) and brackish water (Schaproder Bodden). We identified plant species, plant age, and habitat-specific differences and similarities of the bacterial density and community composition.
MATERIAL &METHODS
Plants. Brackish water samples of Chara aspera and Myriophyllum spicatum were collected on 24 October 2006 in the Schaproder Bodden, east of the Isle of Hiddensee (N 54°27.4627’; E 13°07.5664’). Three plants each were collected by snorkelling in 0.7–1 m depth, stored in artificial brackish water (8‰, the same salinity as in the Bodden; (Blindow et al. 2003)) with 3.5% formaldehyde (final concentration). Freshwater plants were sampled at the southwest shore of the Isle of Reichenau, Lake Constance, near a gravel ridge (N 47°42.247, E 9°02.289). Three replicates each were collected on 6 November 2006 at a depth of 0.7–1.2 m for M. spicatum and 2.5–3 m for C. aspera. The plants were transported in separate sterile tubes to the laboratory, where they were fixed with 3.5% formaldehyde (final concentration). All samples were stored at 4 °C until processing started on 7 November 2006. The plant samples were divided into an upper section of the plants apices, approx. 5 cm long, and a lower section, approximately 5–10 cm of stem length above the sediment.
Biomass and chemical analyses. Myriophyllum spicatum was processed as part of our routine sampling campaign, in which plants are dissected into apices, upper and lower leaves, and stems. For C. aspera chemical analyses, we did not differentiate between upper and lower plant parts. Sub-samples of each plant part were incinerated for 6 h at 550 °C to determine the ash-free dry mass. We measured the carbon, nitrogen, and phosphorus content of all plant samples using standard methods (Choi et al. 2002). The total phenolic content of M. spicatum and C. aspera was determined using a modified Folin–Ciocalteau method (Box 1983). The concentration of non-phenolic compounds interfering with the Folin reagent are <5% in M. spicatum (Choi et al. 2002). Those in C. aspera were determined using a modified polyvinylpyrrolidon method (Gross et al. 1996). The major allelochemical of M. spicatum, tellimagrandin II, was quantified by reverse-phase HPLC (Müller et al. 2007). All measurements
provide settlement surfaces for bacteria. The antibacterial and allelopathically active compounds in Chara spp. (Scheffer et al. 1993, Fisher et al. 1998) are difficult to isolate and were not determined here.
Detachment of biofilm. Plant parts were transferred into sterile 50 ml polypropylene tubes containing 50 ml of formaldehyde (3.7% final concentration) and sodium pyrophosphate (0.1 M Na4P2O2×10 H2O, NaPPi).
The biofilm was detached by ultrasonication for 60 s (Laboson 200 ultrasonic bath, Bender & Hobein), followed by 15 min of vigorous shaking (18.3 Hz, Thermomixer Eppendorf) and again 60 s of ultrasonication. Two millilitres of the detached biofilm were filtered onto white polycarbonate filters (0.2 µm, Ø 25 mm Nucleopore) and stored at –20 °C.
We optimized the detachment procedure prior to this experiment. NaPPi was a suitable detergent to detach bacteria from macrophyte leaves as shown by a previous study in our group (Müller et al. 2007). We further varied the sonication time and shaking duration to obtain the best results for a gentle but effective detachment of the biofilm (Buesing & Gessner 2002, Bockelmann et al.
2003). Detachment with an ultrasonic probe (Bandelin electronic GM 70 HD, 20 kHz, 57W) resulted in 0.13 ± 0.03×106 cells cm-2 but the plant tissue was severely damaged and numerous bacterial cells were still attached to the leaf surface as observed by microscopic examination. We then tried are more gentle detachment with shorter sonication times in an ultrasonic bath and constant, gentle shaking afterwards, rather than permanent ultrasonication. This method yielded 1.9 ± 0.6×106 cells cm-2 and the plant tissue was not visibly damaged except at the cut surface on the petiole. A thorough microscopy of the leaves proved hardly any attached bacterial cells left.
Fluorescence in situ hybridization (FISH). FISH was conducted following a protocol by Pernthaler et al. (Pernthaler et al. 2001) consisting of a hybridization step at 46 °C for 3 h and a washing step for 15 min at 48 °C.
Filters were counterstained with DAPI (4ʹ,6-diamidino-2-phenylindol,
1 µg ml-1, 5 min). Stained cells were counted under an epifluorescence microscope (Labophot 2, Nikon) at an excitation wavelength of 549 nm. Probes used are listed in Table 2.1 and further details are available at probeBase (Loy et al. 2003).
Table 2.1. Oligonucleotide probes used in this study.
Probe a) Sequence %
FA Target group Reference EUB338 GCTGCCTCCCGTAGGAGT 35 Most bacteria (Amann et al. 1990) NON338 ACTCCTACGGGAGGCAGC 35 Competitor of EUB (Wallner et al. 1993) ALF968 GGTAAGGTTCTGCGCGT 20 Alphaproteobacteria (Neef 1997) BET42a b) GCCTTCCCACTTCGTTT 35 Betaproteobacteria (Manz et al. 1992) GAM42ab)
b)
GCCTTCCCACATCGTTT 35 Gammaproteobacteria (Manz et al. 1992) PLA886 b) GCCTTGCGACCATACTCCC 35 Planctomycetes (Neef et al. 1998) HGC96a TATAGTTACCACCGCCGT 25 Actinomycetes (Roller et al. 1994) CF319a TGGTCCGTGTCTCAGTAC 35 Bacteroidetes (Manz et al. 1996)
a) Probes were labelled with Cy 3 b) For these probes, a competitor probe was used; FA:
Formamide
Statistical analyses. Data of FISH analysis were arcsin transformed. For planctomycetes, data were additionally x¼ transformed to ensure equal variances. Plant species-, plant part-, and habitat-specific differences were analysed by 3-way ANOVAs (Sigma STAT 3.0). Non-metric dimensional scaling plots were generated with square-root transformation of data and Bray-Curtis similarity (Primer 5.0). For correlations, the Pearson correlation was used (Sigma STAT 3.0).
RESULTS
The two plant species, each from two different habitats, exhibited distinct morphological and chemical characteristics. The organic content of the plants of each species and from each habitat differed, but the upper or lower parts of each plant sampled did not differ in organic content (Figure 2.1; 3-way ANOVA, Table 2.2). Chara aspera had a lower organic content (40.9% ± 14.4, mean ± SD, n = 12) than Myriophyllum spicatum (51.7% ± 11.6, mean ± SD, n = 12).
Freshwater plants had a much lower organic content (35.1% ± 9.4, mean ± SD, n = 12) than brackish water plants (57.5% ± 6.6, mean ± SD, n = 12).
Only a marginal interaction of plant × habitat was found (p = 0.079), owing to a larger difference in organic content of C. aspera from the two sites than that of M. spicatum. The significant interaction term between habitat and plant part is due to the observed differences of plant parts in Lake Constance; the organic content of the plant parts did not differ in plants from Schaproder Bodden.
SB M upper SB M lower SB C upper SB C lower LC M upper LC M lower LC C upper LC C lower
% organic dry mass
0 20 40 60
80 Figure 2.1 Proportion of organic
dry mass in plant samples collected at all sites.
SB: Schaproder Bodden, LC: Lake Constance, C: Chara aspera, M: Myriophyllum spicatum; upper and lower indicate plant parts analysed; n = 3; error bars indicate SE
Myriophyllum spicatum contained more phenolic compounds than C. aspera [97–173 mg (g dry mass)–1 vs. <1 mg (g dry mass)–1; Table 2.3] and M. spicatum from Lake Constance had a slightly higher polyphenol content than M. spicatum from Schaproder Bodden [apices: 173 ± 21 mg (g dry mass)–1 and 120 ± 33 mg (g dry mass)–1, respectively; Student’s t-test: P = 0.02]. Also the anthocyanin content was much higher in M. spicatum than in C. aspera. In both habitats, the anthocyanin content of C. aspera was <0.1 mg (g dry mass)–1; the anthocyanin content of M. spicatum from Schaproder Bodden was slightly lower than that of M. spicatum from Lake Constance [approx. 0.5 mg (g dry mass)–1 vs. 1.0 mg (g dry mass)–1; Student’s t–test: P = 0.005, Table 2.2]. The chlorophyll a and b contents were highest in the apical shoots and upper leaves of M. spicatum from Lake Constance (Table 2.3).
Table 2.2. Statistical analysis. 3-way ANOVA for selected parameters. Data for the CFB were arcsin transformed; for planctomycetes, x1/4 transformation was used.
% Plant organic matter
Ash-free dry mass
Total bacterial cell
counts
Planctomycetes CFB Source
of varia- tion
DF F P F P F P F P F P
Habitat 1 101.63 <0.001 13.721 0.002 25.963 <0.001 30.970 <0.001 0.467 0.504 Plant 1 24.481 <0.001 3.746 0.071 1.944 0.182 26.623 <0.001 45.454 <0.001 Plant
part (PP)
1 0.0563 0.815 0.183 0.674 21.229 <0.001 3.705 0.072 21.018 <0.001 Habitat
× Plant 1 3.510 0.079 0.0307 0.863 2.606 0.126 10.618 0.005 0.538 0.474 Habitat
× PP 1 5.249 0.036 2.253 0.153 10.499 0.005 0.484 0.497 4.901 0.042 Plant ×
PP 1 1.087 0.313 0.0479 0.830 0.0246 0.877 0.0998 0.756 7.105 0.017 Habitat
× Plant
× PP
1 0.505 0.488 0.121 0.732 < .001 0.995 1.179 0.294 14.113 0.002
The carbon content (Table 2.3) of C. aspera was about half of that of M. spicatum, possibly in part owing to the overall lower organic dry mass of the former. Chara aspera also contained less nitrogen and phosphorus per g dry mass than M. spicatum when whole plants were considered. The C/N molar ratio ranged from about 15 in apices of M. spicatum from Lake Constance to 31 in stems of M. spicatum from Schaproder Bodden. The C/P molar ratio ranged from 436 in apices of M. spicatum to more than 1373 in C. aspera from Lake Constance.
Table 2.3. Chemical parameters measured in plants.
LC: Lake Constance, SB: Schaproder Bodden, n = 3, mean ± SD Total phenolic content
[mg (g dry mass)–1]
Anthocyanin [mg (g dry mass)–1]
Chlorophyll a and b [mg (g dry mass)-–1]
LC SB LC SB LC SB
C. aspera 0.9 ± 0.08 0.7 ± 0.12 0.05 ± 0 0.06 ± 0 1.9 ± 0.17 1.2 ± 0.02 M. spicatum
173 ± 21 120 ± 33 0.9 ± 0.02 0.52 ± 0.08 6.8 ± 1.3 2.3 ± 0.26 120 ± 29 97 ± 5 0.8 ± 0.2 0.50 ± 0.01 8.4 ± 2.3 2.3 ± 0.28 Apex
Upper leaves
Upper stem 133 ± 13 100 ± 9 1.4 ± 0.1 0.81 ± 0.15 1.8 ± 0.6 1.0 ± 0.04 C
[mg (g dry mass)–1]
N
[mg (g dry mass)–1]
P
[mg (g dry mass)–1]
LC SB LC SB LC SB
C. aspera 206 ± 4 179 ± 15 11 ± 1.1 14 ± 0.12 0.4 ± 0.1 0.68 ± 0.06 M. spicatum
425 ± 20 357 ± 35 36 ± 9 24 ± 8 2.6 ± 1.2 2.4 ± 1.5 384 ± 37 363 ± 5 27 ± 6 16 ± 3 1.2 1.3± 0.33 Apex
Upper leaves
Upper stem 400 ± 7 379 ± 25 15 ± 3 14 ± 3 0.9 ± 0.2 ––
We determined the bacterial abundance based on plant dry mass since there are no reliable surface area-to-biomass ratios for M. spicatum and C. aspera from the two habitats. The bacterial abundance in the two habitats and on the different plant parts differed significantly, but did not differ significantly between the two plant species (Figure 2.2, Table 2.2). In general, we found a higher bacterial abundance on plants from Schaproder Bodden [5.1×107 ± 3.9×107 cells (g dry mass)–1; mean ± 1 SD] than on plants from Lake Constance [1.3×107 ± 0.7×107 cells (g dry mass)–1]. The lower plants parts from
Schaproder Bodden had higher bacterial cell counts than the upper plant parts, while cell counts on lower plant parts from Lake Constance were only marginally higher than the counts on upper plant parts (Figure 2.2), resulting in a significant habitat × plant part interaction (Table 2.2, P = 0.005). The ash- free dry mass differed significantly between habitats, and the organic content of the plant samples differed significantly between habitats and plant species but not between plant parts (Table 2.2). The general pattern of bacterial abundance remained when calculated on an organic dry matter basis (Figure 2.2).
SB M upper SB M lower SB C upper SB C lower LC M upper LC M lower LC C upper LC C lower
Total bacterial cell counts (g dm)-1 x 107 0 5 10 15 20 25
Figure 2.2.Total bacterial cell counts determined by DAPI staining. Black bars: counts (g dry mass)–1; grey bars: counts (g ash-free dry mass)–1. SB: Schaproder Bodden; LC: Lake Constance;
M: M. spicatum; C: C. aspera. n = 3; error bars indicate SE.
A)
0 10 20
30 B)
0 10 20 30
H)
ALF BET GAM PLA HGC CFB
0 10 20 30 E)
0 10 20 30
G)
ALF BET GAM PLA HGC CFB
0 10 20 30
F)
0 10 20 30 C)
% of DAPI counts
0 10 20
30 D)
0 10 20 30
The composition of the bacterial biofilm on the two plant species was similar except for the abundance of members of the CFB group and planctomycetes (Figure 2.3). On both plant species in both habitats, bacteria of the CFB group were the most abundant bacterial group and reached up to 35%
of the total cell counts. The CFB counts correlated positively with all measured chemical parameters (Pearson correlation: carbon: r = 0.637, P = 0.0008; nitrogen: r = 0.666, P = 0.0003; phosphorus: r = 0.755, P < 0.0001;
chlorophyll: r = 0.433, P = 0.0344; total phenolic compounds: r = 0.685, P = 0.0002). The number of CFB cells was generally higher on M. spicatum than on C. aspera and higher on upper parts of both plant species. The differences were not uniform and resulted in significant interaction terms (Figure 2.3;
Table 2.2), which indicated specific habitat, plant, and plant part patterns. The
Figure 2.3. Biofilm composition in Lake Constance (left) and Schaproder Bodden (right).
A and B, Myriophyllum spicatum upper section;
C and D, Chara aspera upper section;
E and F, M. spicatum lower section;
G and H, Chara aspera lower section.
n = 3; errors bars indicate SD.
ALF: alphaproteobacteria;
BET: betaproteobacteria;
GAM: gammaproteobacteria;
PLA: planctomycetes;
HGC: actinomycetes;
CFB: Cytophaga–Flavobacteria–
Bacteroidetes
Stress: 0.12
second major group of bacteria in the biofilms were alphaproteobacteria, which accounted for 3–17% of the DAPI counts. The abundance of alphaproteobacteria did not differ between plant species and habitats (3-way ANOVA, df = 1, F = 4.1, P = 0.05). Beta- and gammaproteobacteria abundance was similar on both plant species and in both habitats (3-way ANOVA, df = 1, F = 1.257, P = 0.279; df = 1, F = 1.982, P = 0.178). Actinomycetes were the least- abundant group, and their abundance did not differ between plant species (0.7–2.0% of DAPI counts¸ 3-way ANOVA, df = 1, F = 1.179, P = 0.294).
Figure 2.4. Non-metric dimensional scaling plot of the bacterial community composition on all plant samples.
Grey triangles: Myriophyllum spicatum, white triangles: Chara aspera. Striped
triangles: samples from Schaproder Bodden; non-striped triangles: samples from Lake Constance. Upper and lower plant parts are denoted by triangles pointing upwards and downwards, respectively. Data are x1/4 transformed.
Interestingly, the proportion of planctomycetes differed between habitat and plant species. In Lake Constance, almost no planctomycetes were detected on M. spicatum, but they made up 2–3% of all cell counts on C. aspera. In Schaproder Bodden, planctomycetes were found on both plant species, with slightly higher numbers on the upper plant parts (2–6% of DAPI counts) than
