in Lower Lake Constance
Melanie Hempel & Elisabeth M. Gross
ABSTRACT: The heterotrophic biofilm community composition (BCC) on the aquatic macrophytes Myriophyllum spicatum (MS), Potamogeton perfoliatus (PP) and artificial substrates (Art) was compared on a spatial (plant age) and temporal (season) scale in 2006. If plants were neutral substrates for microorganisms, no differences in BCC compared with the propylene sheets should be observed.
Myriophyllum spicatum contains polyphenolic allelochemicals, which inhibit algae, cyanobacteria and bacteria, and might influence the BCC. Major bacterial groups were identified by FISH (fluorescence in situ hybridization). We also measured plant stoichiometry and content of phenolic compounds. To investigate the initial bacterial colonization, we exposed axenic M. spicatum in an outdoor mesocosm for 72 hrs.
Total bacterial cell counts were highest on the artificial substrate (1.3 ± 0.3×106 cm–2), followed by M. spicatum (0.8 ± 0.3×106 cm–2) and P. perfoliatus (0.3 ± 0.2×106 cm–2), and increased towards autumn. Members of the CFB group (mean on MS 22%, PP 21%, Art 16%), alphaproteobacteria (mean on all substrates 19%) and betaproteobacteria (mean on MS 17%, PP 31%, Art 7%) were always abundant, while actinobacteria and planctomycetes occurred less frequently. Potamogeton perfoliatus had the highest proportion of gammaproteobacteria (average 19%), while on M. spicatum and the artificial substrate the shares were 9% and 4%, respectively. Alpha– and betaproteobacteria displayed a slight seasonality. The initial colonization of axenic M. spicatum was dominated by the CFB group at 5 hrs, followed by betaproteobacteria. Plant chemistry differed substantially between plants, and within M. spicatum. Differences in BCC were mainly explained by environmental factors (water level, conductivity, temperature, pH) and by plant chemistry, especially carbon and total phenolic content.
Keywords: Myriophyllum spicatum, Potamogeton perfoliatus, allelochemical interactions, fluorescence in situ hybridization, phenolic compounds, water level, conductivity
INTRODUCTION
Submerged macrophytes are not only the major primary producers in the littoral zones of lakes they also structure this zone by reducing sediment resuspension, providing spawning area and shelter for young fishes and zooplankton. They further offer a vast surface area for the attachment of various organisms, from bacteria and algae to invertebrates (Jeppesen et al. 1998).
Heterotrophic bacteria largely contribute to the overall nutrient cycling and interact in various ways with other organisms by relocating nutrients, converting degradation products, restoring growth forms of macroalgae, facilitating spore attachment and preventing grazing (Joint et al. 2000, Buesing & Gessner 2006, Marshall et al. 2006). Further, they can invade and damage tissue and promote biofouling (Underwood 1991). Excessive biofilm formation also has potential negative consequences for the host due to decreased exchange of nutrients and reduced photosynthesis (Phillips et al. 1978, Sand-Jensen & Søndergaard 1981). In the root sections of macrophytes, bacteria are generally recognized as important mediators for macrophytes` nutrient uptake, esp. nitrogen (Eriksson & Weisner 1999). In return, macrophytes provide resources for bacteria, e.g., exuded organic compounds or gases like methane from the root zone, which is transported through the lacunar system to the above–ground plant parts and released into the water column (Gross et al. 1996, Schuette 1996, Heilman & Carlton 2001).
Taking these aspects into account, the littoral zone is not solely characterized by the macrophyte community but also by their autotrophic and heterotrophic biofilms.
From terrestrial plants it is known that they often invest in chemical defence against competitors, pathogens and herbivores. A macrophyte with a high allelochemical potential in Lake Constance is Myriophyllum spicatum L. Besides its canopy forming growth, it produces high amounts of hydrolysable polyphenols that retard larval growth and inhibit bacteria and cyanobacteria (Choi et al. 2002, Leu et al. 2002, Walenciak et al. 2002). These polyphenols are not only in the plant tissue but may gradually leak from leaves into the surrounding water. Thus, biofilms
establishing on the surface of these plants are exposed to these compounds in high concentrations, which may result specific adaptations, e.g., to polyphenols, which can be utilized as substrate (Müller et al. 2007). Another macrophyte growing in the vicinity of M. spicatum in Lake Constance is the pondweed Potamogeton perfoliatus. It forms large stands up to the water surface, but does not contain polyphenols, only few other phenolic compounds. Several pondweeds, however, contain diterpenes which inhibit microalgae (DellaGreca et al. 2001), but none of them has been found in P. perfoliatus.
Little attention has been paid to the heterotrophic biofilm on submerged macrophytes, especially to spatial differences between older and younger leaves.
Hence, the knowledge on the bacterial community composition (BCC) of these biofilms is extremely scarce. Molecular studies investigating aquatic surfaces have mostly been on artificial substrates (Brummer et al. 2000, Olapade & Leff 2006), algae (Rao et al. 2006), among them diatoms (Grossart et al. 2005). Others investigated the heterotrophic biofilm on macrophytes with cultivation dependent techniques (Chand et al. 1992), which can be rather selective depending on growth conditions applied.
In this study, we focus on the spatial and temporal community composition of the heterotrophic biofilm on aquatic surfaces. We investigated Myriophyllum spicatum, Potamogeton perfoliatus and an artificial substrate throughout the vegetation period in summer 2006 with fluorescence in situ hybridization (FISH). We distinguished along a vertical axis between plant apices, middle and lower leaves. Plant quality along this gradient was analysed by measuring carbon, nitrogen, phosphorus, chlorophyll and total phenolic content (anthocyanins and tellimagrandin II).
MATERIAL &METHODS
Sampling site. The sampling site (N47°42.247, E9°02.289) is located at the Isle of Reichenau in Lower Lake Constance, Germany, near a gravel ridge. Myriophyllum spicatum, Potamogeton perfoliatus and artificial substrates were taken biweekly in triplicates by snorkelling in a depth of 1.5 – 2 m in the time from 17 July to 09 October 2006. Plants and artificial substrate samples were stored individually in polyethylene tubes at 4 °C until processing. For chemical analysis, plants were stored in plastic bags at 4 °C until analysis the next day. Temperature, oxygen, conductivity and pH were measured in the water column at each sampling date. For a detailed description of the experimental set–up see Hempel et al. (submitted).
Detachment of epiphytic biofilm. In the laboratory, plant length was measured and the overall state of the plant was recorded. Artificial substrates were documented through photography. Each one leaf of the apex, one leaf of the 1 – 10 cm (middle leaf) and 10 – 25 cm (lower leaf) shoot section were taken from three plants originating from three different stands and these nine leaves were transferred to 1 ml sodium pyrophosphate (0.1 M Na4P2O7×10 H2O) containing 3.7% formaldehyde. Of the artificial substrates, one section of approx. 1 cm2 was cut off and also transferred into 1 ml sodium pyrophosphate. The detachment of the biofilm had been optimized and was conducted as described in (Hempel et al. 2008).
After detachment, leaves were transferred into one millilitre of tap water and stored at 4 °C until leaf surface was measured. The detached biofilm was filtered onto white polycarbonate filters (0.2 µm; Ø 25 mm, Schleicher & Schuell) and stored at –20 °C.
Fluorescence in situ hybridization (FISH). FISH was performed following a protocol consisting of a hybridization step at 46 °C for three hours and a washing step for 15 min at 48 °C (Pernthaler et al. 2001). Filters were counterstained with DAPI (4’,6–Diamidino–2–phenylindol, 1 µg ml–1, 5 min). Stained cells were counted under an epifluorescence microscope (Labophot 2, Nikon) at an excitation of 549 nm and 1000× magnification. The probes used are listed in Table 4.1, and further details are available at probeBase (Loy et al. 2003).
Table 1. Oligonucleotide probes used in this study.
Probe a) Sequence % Form–
amide Target group Reference
EUB338 GCTGCCTCCCGTAGGAGT 35 Most Bacteria (Amann et al. 1990) NON338 ACTCCTACGGGAGGCAGC 35 Competitor to EUB (Wallner et al. 1993) ALF968 GGTAAGGTTCTGCGCGT 20 Alphaproteobacteria (Neef 1997) BET42a b) GCCTTCCCACTTCGTTT 35 Betaproteobacteria (Manz et al. 1992) GAM42a 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
Measurement of leaf surface. To relate total cell counts to the surface area of the plants, leaves were photographed with a Nikon D70S and analysed with
“Makrophyt” a computational program designed by the scientific workshops of the University Constance. Each leaf was photographed with three different exposure times and the mean leaf size calculated. The calculated area of M. spicatum was multiplied by π (3.14) to account for the circular shape of the leaves. To calculate the leaf surface of P. perfoliatus, the area was multiplied by two since the oval leaves are laminar.
Chemical analysis. Different plant parts were analysed by spectrophotometry for total phenolic content (Folin–Ciocalteau assay; (Box 1983)), anthocyanin (Murray &
Hackett 1991), carbon, nitrogen, phosphorus (Choi et al. 2002), chlorophyll a and b (Porra 1990) content and for M. spicatum only, by HPLC for tellimagrandin II (Müller et al. 2007). Folin–sensitive compounds in P. perfoliatus are only to 50% phenolic compounds (Choi et al. 2002), thus the Folin–results were halved to reflect the true total phenolic content. Myriophyllum spicatum was part of our routine sampling campaign, and three replicates resulting from three different stands were measured.
We measured P. perfoliatus plants originating from one stand, and thus only one measurement for each sampling date is available. We experienced that plants originating from one location usually do not differ substantially in chemical composition.
Initial colonization of axenic M. spicatum. Axenic M. spicatum rooted plants of ca.
8 cm length were planted into an outdoor concrete basin (2×2×1 m) in the yard of the Limnological Institute in May 2007. The plants were anchored into the sediment by tying them to stainless steel screw–nuts. After 5, 24, 36, 48 and 72 hours, three leaves from three different plants were taken and analyzed for bacterial community composition with FISH. Detachment of bacteria and FISH were performed as described above.
Statistics. To analyse significant differences and potential interactions between the biofilm community composition on the substrates at different times, we used one–
way ANOVAs to compare differences between all three substrates or between individual sampling dates, Mann–Whitney rank sum tests to distinguish differences between parts of both plants and Pearson correlations to investigate continuous seasonal changes (Sigma Stat 3.11, Systat Software, Inc.). The proportional FISH data were arcsin transformed, and data for gammaproteobacteria, planctomycetes, actinomycetes and CFB were additionally x1/4 transformed to yield equal variance. To account for the multiple comparisons, we set our level of significance at α = 0.01.
We related the FISH abundance data to plant chemistry and environmental conditions with a BEST–ENV analysis to see which factors explain the differences between both plant species best. A dissimilarity matrix was calculated based on Bray Curtis dissimilarity for square root transformed FISH data, and a dissimilarity matrix was calculated for standardised environmental data with Euclidean distance as described before (Hempel et al., submitted). For the plant chemistry, we chose tissue nitrogen, carbon, phosphorus, chlorophyll and total phenolic content and as environmental factors water level, temperature, conductivity and pH. The analyses were performed with Primer 6 (Version 6.1.6, Primer E Ltd.).
RESULTS
Initial colonization of axenic Myriophyllum spicatum
Total bacterial cell counts on freshly colonized M. spicatum were in the same range as data from the lake sampling site or even higher (0.75 – 2.3×106 cells cm–2), thus plants are colonized very rapidly within 5 hours (Figure 4.1A). A distinct succession of bacterial groups was observed (Figure 4.1B). In the first five hours, members of the CFB–group made up the major portions of the biofilm (46% of DAPI counts), followed by betaproteobacteria (24% of DAPI counts) and actinomycetes (6% of DAPI counts). After 24 h, the biofilm was dominated by betaproteobacteria (56 – 62%
of DAPI counts), followed by members of the CFB–group. Alpha– and gammaproteobacteria increased steadily at low numbers to a maximum of 4% and 5% of total cell counts, respectively.
Figure 4.1: Initial colonization of axenic Myriophyllum spicatum in a mesocosm over 72 hours.
A) Total bacterial cell counts over the sampling duration
B) Bacterial biofilm community composition as determined by FISH.
Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Actinobacteria
Planctomycetes CFB-Phlyum
Total bacterial cell counts
On M. spicatum, we did not observe any significant influence of plant age or season on the total bacterial cell counts. On the apices, cell counts (0.63 ± 0.24×106 cells cm–2) were in the range to that on middle leaves (0.66 ± 0.1×106 cells cm–2), and both were slightly lower than on lower leaves (1 ± 0.31×106 cells cm–2). Towards autumn, a slight increase in total bacterial cell counts on the lower leaves was monitored (Figure 4.2A). On Potamogeton perfoliatus, total bacterial cell counts were rather similar on the different plant parts throughout the season (apex: 0.43 ± 0.31×106, middle leaves: 0.2 ± 0.1×106; lower leaves:
0.28 ± 0.13×106 cells cm–2) with slightly higher cell counts for the apices (Figure 4.2B).
We observed a marked rise on 12 September (0.6 ± 0.3×106 cells cm–2) and a subsequent decline afterwards. The artificial substrates had about 13–fold higher cell counts at the end of the sampling campaign than at the beginning (from 0.3± 0.03×106 to 5 ± 3×106 cells cm–2). With values of 1.78 ± 1.5×106 cells cm–2 over the whole season, the bacterial numbers were higher on the artificial substrate than on both macrophytes (Figure 4.2C). The bacterial cell counts on P. perfoliatus were lower than those on M. spicatum, especially between the middle and lower leaves (Mann-Whitney rank sum test, middle and lower leaves both P < 0.001).
Figure 4.2. Total cell counts on all substrates in the vegetated period.
A) Myriophyllum spicatum B) Potamogeton perfoliatus C) Artificial substrate. White circles: Apex; triangle up: Middle leaves; triangle down: lower leaves. Note that the y–axis in 2C is differently scaled.
A
Bacterial community composition
The bacterial community composition was assessed by FISH (fluorescence in situ hybridization). Usually, eubacterial counts were above 50% of DAPI counts (65% ± 16, mean ± SD, for all dates and substrates). On all substrates, bacteria of the CFB and beta– and alphaproteobacteria were abundant, followed by gammaproteobacteria, while actinomycetes and planctomycetes were of minor importance (Figure 4.3).
Spatial and temporal variability on different substrates. On M. spicatum, the biofilm community composition did not differ strongly between sampling dates or plant parts (Figure 4.3A, C and E). In general, proportions of CFB and betaproteobacteria were high throughout the vegetation period and ranged between 3 – 75% and 3 – 58% of DAPI counts, respectively, and no seasonal trends were observed. In a few cases, the hybridization efficiency was low, and no CFB bacteria could be detected, which might be caused by the low hybridization efficiency of less than 50% of this probe (see Figure 4.3).
The apices of M. spicatum had the highest CFB shares (32 ± 17% of DAPI counts) followed by the middle (16 ± 12% of DAPI counts) and lower leaves (15 ± 10% of DAPI counts). Alphaproteobacteria increased on the apices during the season (2 – 43% of DAPI counts, Pearson correlation P < 0.01), stayed more or less constant on middle leaves (18 ± 7% of DAPI counts, Pearson correlation P = 0.761) and decreased on the lower leaves towards fall (from 46% to 10% of DAPI counts, Pearson correlation P = 0.0155). Planctomycetes and actinomycetes accounted together for 13% of the DAPI counts.
On P. perfoliatus, differences in biofilm community composition between different plant parts were even less pronounced (Figure 3B, D and F). Betaproteobacteria on the leaves doubled from July (13 ± 9% of DAPI counts) to September (52 ± 5% of DAPI counts: One–way–ANOVA, DF = 6, F = 6.25, P < 0.001, Holm Sidak post hoc test P < 0.005 for comparisons between July and September).
E
Figure 4.3: Biofilm community composition on Myriophyllum spicatum, Potamogeton perfoliatus and artificial substrates. A) M. spicatum apex B) P. perfoliatus apex C) M. spicatum middle leaf D) P.
perfoliatus middle leaf E) M. spicatum lower leaf F) P. perfoliatus lower leaf G) Artificial substrate. n = 3.
The standard deviation was between 7 – 135% and is not displayed for more visibility
Bacteria belonging to the CFB–group made up the largest portion of all detected bacteria on all plant parts (range 10 – 50% of DAPI counts, Figure 3B, D and F). In general, CFB counts on all plant parts declined towards fall with an intermediate peak in mid August (54 ± 21% of DAPI counts). Alphaproteobacteria ranged from 8 – 27% of DAPI counts on all plant parts, and exhibited no seasonal trend with the exception of the apices, where their numbers increased (Pearson correlation P = 0.0186), comparable to M. spicatum.
The biofilm on artificial substrates was dominated by alphaproteobacteria (23 ± 10% of DAPI counts), CFB (16 ± 10% of DAPI counts) and betaproteobacteria (8% of DAPI counts, Figure 4.3G). Gammaproteobacteria, planctomycetes and actinomycetes accounted together for up to 10% of the biofilm community. We did not find any seasonal trend for all bacterial groups on this substrate.
Comparison between substrates. Potamogeton perfoliatus had the highest proportions of betaproteobacteria, especially on the middle and lower leaves compared to the respective M. spicatum parts (t– test, P = 0.08 and P < 0.001, respectively), and both P. perfoliatus parts had much higher counts than M. spicatum and the artificial substrates (17 ± 8% of DAPI counts for M. spicatum, 31 ± 12% of DAPI counts for P. perfoliatus and 7 ± 8% of DAPI counts for artificial substrates, one–way ANOVA, Holm Sidak post hoc test P < 0.0001 for both comparisons). CFB and alphaproteobacteria did not differ between plants, irrespective of plant part, and were found in the same range (16 – 20% of DAPI counts) on the artificial substrates.
Gammaproteobacteria were higher on P. perfoliatus than on M. spicatum on every plant part (19 ± 10% and 9 ± 4% of DAPI counts, respectively; Mann–Whitney rank sum test P < 0.001). Gammaproteobacteria on the artificial substrate were present on all sampling dates but in rather variable numbers (1 – 11% of DAPI counts).
Actinomycetes were present in low but consistent numbers on all substrates, ranging between 1 – 22% of DAPI counts but we did not find any effect of the substrate nor the plant age. Planctomycetes occurred more often on M. spicatum, and there at times in rather high abundance of up to 29% of DAPI counts on the apices, but they were
also lacking frequently. On P. perfoliatus, planctomycetes were rather absent throughout the vegetation period, while we found constant but low numbers on the artificial substrates.
Chemical analysis
Plant stoichiometry. In M. spicatum, the molar C/N ratio ranged between 15 and 21 and was highly variable during the season and between plant parts. We observed both a gradient from apices (lowest) to lower leaves (highest), and additionally a decline with season (Figure 4.4A). The seasonal change was due to a rise in nitrogen in the plants [apices 19 – 44 mg (g dm)–1, middle leaf 12 – 36 mg (g dm)–1and lower leaf 7 – 25 mg (g dm)–1] and differences in carbon content [apices 403 – 455 mg (g dm)–1, middle leaf 289 – 437 mg (g dm)–1and lower leaf 201 – 350 mg (g dm)–1]. The molar C/N ratio in P. perfoliatus ranged from 10 to 27, and was more constant throughout the season in leaves compared to the apices (Figure 4.4B). Phosphorus concentration was highest in M. spicatum apices [1.8 to 3.5 mg (g dm)–1] with a rise in autumn. Also in the middle and lower leaves phosphorus increased with season, and the content was slightly higher in lower leaves compared to middle leaves [0.8 ± 0.3 and 0.5 ± 0.2 mg (g dm)–1, respectively, mean ± SD; Figure 4.4C]. In P. perfoliatus, the phosphorus content was very similar in apices and leaves [0.5 to 1.2 mg (g dm)–1], with higher values in mid September and end of October in all plant parts (Figure 4.4D). The chlorophyll content in P. perfoliatus was slightly higher than in M. spicatum (Figure 4E and F), with a strong decrease from the beginning of September until the end of the sampling campaign. The apices [6 ± 2 mg (g dm–1)]
always contained less chlorophyll than the middle and lower leaves [7 ± 2 and 9 ± 3 mg (g dm–1), respectively]. In M. spicatum, chlorophyll content increased in all plant parts with season, and was higher in apices and middle leaves than in lower leaves [5 ± 1, 6 ± 2 and 4 ± 2 mg (g dm–1), respectively; Figure 4.4E].
A) Chlorophyll a & b [mg (g dm)-1 ]
0
Figure 4.4: Chemical parameters of Myriophyllum spicatum (left) and Potamogeton perfoliatus (right). Circles: Apex; triangle up: Middle leaf; triangle down: Lower leaf. n = 3, mean ± SD.
Phenolic compounds. The total phenolic content was highest in M. spicatum apices [200 – 250 mg (g dm)–1], followed by middle and lower leaves [67 – 138 and 50 – 70 mg (g dm)–1, respectively; Figure 4.5A]. Potamogeton perfoliatus contained only 21 ± 9 mg (g dm)–1 of total phenolics, and no difference occurred between apices and leaves (Figure 4.5B).
The anthocyanin content was higher in M. spicatum apices [1.5 ± 0.3 – 2 mg (g dm)-1] than in both leaf sections [0.6 ± 0.3 mg (g dm)–1; Figure 4.5C]. In P. perfoliatus, the anthocyanin content was rather similar between all plant parts [average 0.3 ± 0.07 mg (g dm)–1], and no seasonal variation was observed (Figure 4.5D). Tellimagrandin II is the major hydrolysable polyphenol in M. spicatum and does not occur in P. perfoliatus. In the apices of M. spicatum, the tellimagrandin II content was higher [30 – 70 mg (g dm)–1] than in both leaf sections [2 –20 mg (g dm)–1; Figure 4.5E].
Table 4.2. Environmental variables measured at the sampling dates. Water level data are of the water gauge at the harbour in Konstanz.
Sampling
Date No. sampling date Temperature
[°C] Water level
Impact of plant chemistry and environmental factors on community composition We performed a BEST–ENV analysis to elucidate the major factors influencing bacterial community composition. The analysis indicated that of all plant chemistry parameters measured, only carbon and total phenolic content marginally explained the variation in bacterial community composition between the two plant species (ρ = 0.175, P = 0.1, n = 42).
C)
Anthocyanin [mg (g dm)-1 ]
0 1 2
A)
TPC [mg (g dm)-1 ]
0 100 200 300
E)
Aug Sep Oct Tellimagrandin II [mg (g dm)-1 ]
0 20 40 60
D)
Aug Sep Oct
0 1 2 B)
0 100 200 300
Figure 4.5: Concentration of different phenolic compounds in Myriophyllum spicatum (left) and Potamogeton perfoliatus (right). Circles: Apex; triangle up: Middle leaf; triangle down: Lower leaf. n
= 3 mean ± SD. Tellimagrandin II or hydrolysable polyphenols are not present in P. perfoliatus.
Apex
The environmental factors water level and conductivity explained most of the variability of the biofilm community composition (ρ = 0.33, P = 0.002, n = 42). If both plant chemistry and environmental variables were considered, carbon, total phenolic content, water temperature, water level, conductivity and pH were the major predictors, and the correlation coefficient increased (ρ = 0.354, P = 0.009). To compare the biofilm on the artificial substrates with that on the plants, we carried out the BEST–ENV analysis only with environmental variables. Here, conductivity explained most of the variability in the biofilm community composition (ρ = 0.217, P = 0.002, n = 49).
Despite these differences, the overall bacterial community composition did not
Despite these differences, the overall bacterial community composition did not