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
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
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